T-bet compositions and methods of use thereof

ABSTRACT

Isolated nucleic acid molecules encoding T-bet, and isolated T-bet polypeptides, are provided. The invention further provides antisense nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals carrying a T-bet transgene. The invention further provides T-bet fusion proteins and anti-T-bet antibodies. Methods of using the T-bet compositions of the invention are also disclosed, including methods for detecting T-bet expression and/or activity in a biological sample, methods of modulating T-bet expression and/or activity in a cell, and methods for identifying agents that modulate the expression and/or activity of T-bet.

RELATED APPLICATIONS

This application is continuation-in-part application of U.S. applicationSer. No. 10/008,264, filed on Dec. 3, 2001 (pending), which is acontinuation-in-part application of PCT/US00/15345, filed on Jun. 1,2000 (pending), published pursuant to PCT Article 21, in English, whichclaims priority to U.S. Provisional Application Ser. No. 06/137,085,filed Jun. 2, 1999, the entire contents of each of these applications isincorporated herein by this reference.

GOVERNMENT FUNDING

Work described herein was supported, at least in part, under grantsAI/AG 37833, AI 39646, AI 36535, AR 6-2227, TGAI 07290 awarded by theNational Institutes of Health. The U.S. government therefore may havecertain rights in this invention.

BACKGROUND OF THE INVENTION

Cells of the immune system alter patterns of gene expression in responseto extracellular and intracellular signals. A group of polypeptides,designated cytokines or lymphokines, which affect a range of biologicalactivities in several cell types, are among the most important of thesesignals. While many cell types in the immune system secrete cytokines,the T helper (Th) lymphocyte is the major source of these polypeptides.More than a decade ago it was discovered that Th cells differentiateinto two distinct subsets, Th1 and Th2, upon T cell receptor engagement,defined both by their distinct functional abilities and by uniquecytokine profiles (Paul and Seder, 1994, Cell 76, 241-251; Mosmann andCoffman, 1989, Annu. Rev. Immunol. 7, 145-173; Mosmann et al., 1986, J.Immunol. 136, 2348-2357; Snapper and Paul, 1987, Science 236, 944-947).Th1 cells mediate delayed type hypersensitivity responses and macrophageactivation while Th2 cells provide help to B cells and are critical inthe allergic response (Mosmann and Coffman, 1989, Annu. Rev. Immunol. 7,145-173; Paul and Seder, 1994, Cell 76, 241-251; Arthur and Mason, 1986,J. Exp. Med. 163, 774-786; Paliard et al., 1988, J. Immunol. 141,849-855; Finkelman et al, 1988, J. Immunol. 141, 2335-2341). Theevidence that Th1 cells directed cell-mediated immunity while Th2 cellscontributed to humoral responses fit nicely with the observations thatan organism tends to mount either a cell-mediated or humoral response,but not both, in response to pathogens. These functional differencesbetween the Th subsets can be explained most easily by the activities ofthe cytokines themselves. IFN-γ is the “signature” cytokine of Th1 cellsalthough Th1 cells also produce IL-2, TNF and LT. The corresponding“signature” cytokine for Th2 cells is IL-4. Th2 cells also secrete IL-5,IL-6, IL-9, IL-10 and IL-13.

Upon encountering antigen, the naive CD4+ T helper precursor (Thp) cellenacts a genetic program that ultimately sends it down a Th1 or Th2lineage. While it is clear that polarization can be achieved bymanipulating the antigen and costimulatory signals i.e. the “strength ofsignal” received by the Thp (Constant and Bottomly, 1997. Annu. Rev.Immunol. 15, 297-322), the most potent inducers of effector Th cells areundoubtedly the cytokines themselves. IL-4 promotes Th2 differentiationand simultaneously blocks Th1 development, an effect that is mediatedvia the Stat6 signaling pathway. Thus, mice that lack IL-4 or Stat6,fail to develop Th2 cells (Kopf et al., 1993, Nature 362, 245-248; Kuhnet al., 1991, Science 254, 707-710; Kaplan et al., 1996, Immunity 4,313-319; Shimoda et al., 1996, Nature 380, 630-633; Takeda et al., 1996,Nature 380, 627-630). In contrast, IL-12, IL-18 and IFN-γ are thecytokines critical for the development of Th1 cells (Hsieh et al., 1993,Science 260, 547-549; Okamura et al., 1995, nature 378, 88-91; Gu etal., 1997, Science 275, 206-209; Meraz et al., 1996, Cell 84, 431-442;Magram et al., 1996, Immunity 4, 471-481). IFN-γ acting via the Stat1pathway (Meraz et al., 1996, Cell 84, 431-442), and IL-12, acting viathe Stat-4 signaling pathway (Jacobson et al., 1995, J. Exp. Med. 181,1755-1762) together promote the differentiation of Th1 cells and blockcommitment to the Th2 lineage (Szabo et al., 1995, Immunity 2, 665-675;Szabo et al., 1997, J. Exp. Med. 185: 817-824). Mice deficient in IL-12or Stat4 do not have Th1 cells (Magram et al., 1996, Immunity 4,471-481; Takeda et al., 1996, Nature 380, 627-630; Shimoda et al., 1996,Nature 380, 630-633). Another important Th1-inducing cytokine is IL-18,whose receptor is related to the IL-1 receptor family (Cerretti et al.,1992, Science 256, 97-100). Mice lacking IL-18 have defective in vivoTh1 responses (Takeda et al., 1998, Immunity 8, 383-390) and both IL-12and IL-18 regulate IFN-γ expression (Barbulescu et al., 1998, Eur. J.Immunol. 27, 1098-1107; Robinson et al., 1997, Immunity 7, 571-581; Ahnet al., 1997, J. Immunol. 159, 2125-2131). The cytokines themselves,then, form a positive and negative feedback system that drives Thpolarization (Powrie and Coffman, 1993, Immunol. Today 14, 270-274;Scott, 1991, J. Immunol. 147, 3149; Maggi et al., 1992, J. Immunol. 148,2142; Parronchi et al., 1992, J. Immunol. 149, 2977; Fargeas et al.,1992, Eur. J. Immunol. 149, 2977; Manetti et al., 1993, J. Exp. Med.177, 1199; Trinchieri, 1993, Immunol. Today 14, 335-338; Macatonia etal., 1993, Immunol. 5, 1119; Seder et al., 1993, Proc. Natl. Acad. Sci.USA 90, 10188-10192; Wu et al., 1993, J. Immunol. 151, 1938; Hsieh etal., 1993, Science 260, 547-549) (reviewed in (Seder and Paul, 1994, InAnnual Review of Immunology, Vol. 12, 635-673; Paul and Seder, 1994,Cell 76, 241-251; O'Garra, 1998, Immunity 8, 275-283).

Over the last few years, significant progress has been made inidentifying the transcription factors that control the transition of aThp to a Th2 cell as evidenced by the capacity of such factors to driveIL-4 production reviewed in (Glimcher and Singh, 1999 Cell 96, 13-23;Szabo et al., 1997, Current Opinions in Immunology 9, 776-781). Theprovision of three distinct proteins, the c-Maf proto-oncogene, thetranscription factor Nuclear Factor of Activated T cells (NFAT), and anovel nuclear antigen, NFAT-Interacting Protein 45 kD (NIP45), have beenshown to confer on a non-T cell the ability to produce endogenous IL-4(Hodge et al., 1996, Science 274, 1903-1905; Ho et al., 1998, J. Exp.Med. 188:1859-1866). These factors and others such as GATA-3 (Zheng andFlavell, 1997, Cell 89, 587-596) and Stat6 clearly can drive theproduction of IL-4, and therefore the development of Th2 cells, both invitro and in vivo.

In contrast, little is known about the molecular basis of Th1differentiation. For example, the only known transcription factors whoseabsence results in a failure to generate Th1 cells are Stat4(Thierfelder et al., 1996, Nature 382, 171-174; Kaplan et al., 1996,Nature 382, 174-177) and IRF-1 (Lohoff et al., 1997, Immunity:681-689;Taki et al., 1997, Immunity 6:673-679), neither of which isTh1-specific. The Ets family member ERM which is induced by IL-12 in aStat4-dependent manner has recently been reported to be Th1-specific butit does not affect the production of Th1 cytokines (Ouyang et al, 1999,Proc. Natl. Acad. Sci. 96:3888). The absence of Th1 cells in Stat4deficient mice is secondary to the failure of IL-12 to drive the Th1program while the lack of Th1 cells in IRF-1 deficient mice is likelydue to its direct effect in controlling transcription of the IL-12 gene(Lohoff et al., 1997, Immunity 6: 681-689; Taki et al, 1997, Immunity6:673-679). However, some of the signaling pathways upstream of suchputative Th1-specific regulatory factors are beginning to be elucidated.The p38 kinase is one such signaling molecule as demonstrated by theability of constitutively activated MAP kinase kinase 6 (MKK6) to boostIFN-γ production. Conversely, overexpression of a dominant negative p38MAP kinase or targeted disruption of Jnk2 or Jnk1 reduces Th1 responses(Rincón et al., 1998, EMBO J. 17, 2817-2829; Yang et al, 1998, Immunity9, 575-585; Dong et al., 1998, Science 282, 2092-2095). The JNKsignaling pathway might affect Th development by a direct effect on thetranscription of the IFN-γ gene, but this has not been shown. Forexample, the ATF-2 and AP-1 transcription factors are both substrates ofJNK kinases and these factors as well as NFκB and Stat4 proteins areknown to bind to sites in the IFN-γ promoter (Zhang et al, 1998,Immunol. 161, 6105-6112; Ye et al., 1996, Mol. Cell. Biol. 16:4744;Barbulescu et al., 1997, Eur. J. Immunol. 27, 1098-1107; Sica et al.,1997, J. Biol. Chem. 272, 30412-30420). The production of IFN-γ is,however, normal in mice lacking ATF-2. Because cytokines are critical inthe development of Th1 and Th2 cells and, thereby, in determiningwhether an immune response will be primarily cellular or humoral,compositions and methods for modulating the production of Th1 and/or Th2cytokines would be of tremendous benefit in modulating the immuneresponse.

SUMMARY OF THE INVENTION

This invention is based, at least in part, on the discovery of novelcompositions which act to promote the Th1 phenotype in naïve T helperprecursor cells (Thp), both by initiating Th1 cell genetic programs andby repressing the opposing programs in Th2 cells. In particular, thisinvention provides isolated nucleic acid molecules encoding T-bet andisolated T-bet protein. T-bet (T box expressed in T cells) is a newmember of the T box family of transcription factors whose foundingmember is the brachyury gene. T-bet is constitutively expressedselectively in thymocytes and Th1 cells. T-bet is the first Th1 specifictranscription factor that can transactivate the interferon-γ gene,induce interferon-γ production in retrovirally transduced primary Tcells and redirect polarized Th2 cells into the Th1 pathway. T-bet alsocontrols IFN-γ production in CD8+ T cells, as well as in cells of theinnate immune system, e.g., NK cells and dendritic cells. The inventionalso provides methods of using these novel T-bet compositions.

One aspect of the invention pertains to isolated nucleic acid moleculescomprising a nucleotide sequence encoding a T-bet polypeptide. In oneembodiment, the nucleic acid molecule encodes the polypeptide shown inSEQ ID NO:2 or SEQ ID NO:4. In another embodiment, the nucleic acidmolecule comprises the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO;5 or SEQ ID NO:3. In other embodiments, the nucleic acid molecule hasat least 70% nucleotide identity with at least about 700 contiguousnucleotides of SEQ ID NO:1; at least 70% nucleotide identity with atleast about 500 contiguous nucleotides of SEQ ID NO:3; at least 90%nucleotide identity with at least about 700 contiguous nucleotides ofSEQ ID NO:1; or at least 90% nucleotide identity with at least about 500contiguous nucleotides of SEQ ID NO:3.

In another aspect, the invention pertains to a isolated nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide thatmodulates IFN-γ production in a cell, wherein the nucleotide sequencehybridizes to the complement of the nucleotide sequence set forth in SEQID NO:1 or SEQ ID NO:3 in 6×SSC at 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. In a particular embodiment, thenucleic acid molecule comprises a nucleotide sequence that hybridizesunder conditions of 6×SSC at 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. to at least nucleotides 138-327 of SEQ IDNO:1, or a complement thereof, wherein the nucleotide sequence detects anucleotide sequence encoding a T-bet polypeptide.

The invention also pertains to an isolated nucleic acid molecule whichis antisense to the coding strand of the nucleic acid comprising anucleotide sequence encoding a T-bet polypeptide. The invention furtherpertains to an isolated nucleic acid molecule comprising a nucleotidesequence complementary to mRNA transcribed from a gene encoding a T-betpolypeptide as well as an isolated nucleic acid molecule comprising anucleotide sequence from a gene encoding a T-bet polypeptide, whereinthe nucleotide sequence is not translated. In certain embodiments, thesenucleic acid molecules have a nucleotide sequence between, for example,20-30 or 22-25, nucleotides in length. The nucleic acid molecules of theinvention may be double stranded or single stranded. The invention alsopertains to vectors comprising a nucleotide sequence encoding a T-betpolypeptide, a vector comprising a nucleotide sequence encoding a T-betpolypeptide, e.g., an expression vector, and a host cell containing thevector.

The invention further pertains to a method for producing a T-betpolypeptide comprising culturing a host cell with a vector comprising anucleotide sequence encoding a T-bet polypeptide in a suitable mediumuntil a T-bet polypeptide is produced. The method further comprises thestep of isolating the T-bet polypeptide from the medium or the hostcell.

In another aspect, the invention pertains to isolated T-betpolypeptides. In one embodiment, the T-bet polypeptide comprises theamino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:1or SEQ ID NO:3, or the amino acid sequence of SEQ ID NO: 2 or SEQ IDNO:4. In another embodiment, the T-bet polypeptide comprises at least70% amino acid identity with the polypeptide shown in SEQ ID NO:2 or SEQID NO: 4 and binds to DNA. In yet another embodiment, the T-betpolypeptide has at least 90% amino acid identity with the polypeptideshown in SEQ ID NO:2. The invention also pertains to a fusionpolypeptide comprising a T-bet polypeptide operatively linked to apolypeptide other than T-bet. In one embodiment, the polypeptide otherthan T-bet is the repressor domain of the Drosophila engrailedpolypeptide.

The invention further pertains to antibodies that specifically bind atleast one mammalian T-bet polypeptide. In a preferred embodiment, theantibody specifically binds human T-bet polypeptide. In anotherembodiment, the antibody specifically binds both human and mouse T-betpolypeptide. The antibody can be polyclonal or monoclonal. The antibodymay also be coupled to a detectable substance. Methods of producingantibodies are also encompassed by the invention. For example, a methodof producing an antibody that specifically binds to T-bet comprising,immunizing an animal with a T-bet polypeptide, or antigenic fragmentthereof, such that antibodies are produced by B cells of the animal;isolating B cells of the animal; and fusing the B cells with myelomacells to form immortal, hybridoma cells that secrete human monoclonalantibodies which specifically bind to T-bet, can be performed. Thesubject in these methods can be a non-human transgenic animal having agenome comprising a human heavy chain transgene and a human light chaintransgene. In one embodiment, the method includes a T-bet polypeptide,or a fragment thereof, which comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO: 2 or 4. The invention alsopertains to intrabodies, e.g., an intrabody that specifically binds atleast one mammalian T-bet polypeptide, e.g., an intrabody thatspecifically binds human T-bet polypeptide.

In yet another aspect, the invention pertains to a compositioncomprising an agent that modulates T-bet and a pharmaceuticallyacceptable carrier. The agent may be an agonist of T-bet or anantagonist of T-bet. The agent can modulate the expression and/oractivity of T-bet and an antigen, e.g., the agent can increase theexpression and/or activity of T-bet or the agent decreases theexpression and/or activity of T-bet. The agent can also be a DNAmolecule encoding a T-bet polypeptide; a T-bet polypeptide; or a DNAmolecule encoding a dominant negative T-bet polypeptide. In oneembodiment, the invention pertains to a method for treating a conditionor a disorder that would benefit from modulation of the expressionand/or activity of T-bet in a subject comprising administering theimmunomodulatory compositions described above. The disorder to betreated may be an autoimmune disorder.

In a further aspect, the invention pertains to a nonhuman transgenicanimal, e.g., a mouse, that contains cells carrying a transgene encodinga T-bet polypeptide. The nonhuman animal may comprise in its genome anexogenous DNA molecule that functionally disrupts a T-bet gene of thenon-human animal, wherein the animal exhibits a phenotype characterizedby decreased IFN-γ production, relative to a wild-type animal.

The invention also pertains to a recombinant cell comprising anexogenous T-bet molecule or a portion thereof, and a reporter geneoperably linked to a regulatory region responsive to T-bet such thatupon activation of T-bet, transcription of the reporter gene occurs. Theinvention further pertains to a recombinant cell comprising a geneencoding an ecdysone receptor, an exogenous gene encoding a T-betexpression plasmid operatively linked to the ecdysone gene, and areporter gene operatively linked to an IFN-γ promoter.

The invention also pertains to a method for detecting the presence ofT-bet in a biological sample comprising contacting the biological samplewith an agent capable of detecting an indicator of T-bet activity orexpression such that the presence of T-bet is detected in the biologicalsample. The invention further pertains to a method for modulatingexpression in a cell of a gene whose transcription is regulated byT-bet, comprising contacting the cell with an agent that modulates theexpression or activity of T-bet such that expression of the gene isaltered. In one embodiment, the expression of T-bet is selectivelymodulated. In other embodiments, the agent increases the expressionand/or activity of T-bet or decreases the expression and/or activity ofT-bet. The step of contacting can occur in vivo or in vitro. The cellmay be a T cell, e.g., a lamina propria T cell, a CD8+ cell, and a CD4+cell, a B cell, a dendritic cell, or NK cell. In one embodiment the cellis a mammalian cell, e.g., a human cell. In another embodiment, the geneis a cytokine gene, e.g., IL-2, IFN-γ, IL-4, IL-5, TNFα, TGF-β, LT(lymphotoxin), and IL-10. In yet another embodiment, the gene is acytokine receptor gene

The invention further pertains to a method for modulating the effects ofat least one external influence on a cell, wherein the externalinfluence modulates T-bet expression and/or activity, the methodcomprising contacting a cell with an agent that modulates T-betexpression and/or activity in the cell such that T-bet mediated effectsof at least one external influence is modulated. In one embodiment, theat least one external influence is a cytokine. In another embodiment,the cell is present in a subject suffering from a disorder that wouldbenefit from modulation of T-bet expression and/or activity. In otherembodiments, the external influence is a cytokine the agent increasesthe expression and/or activity of T-bet or the external influence is acytokine the agent decreases the expression and/or activity of T-bet.The cell may be a T cell, e.g., a lamina propria T cell, a CD8+ cell,and a CD4+ cell, a B cell, a dendritic cell, or NK cell. In oneembodiment the cell is a mammalian cell, e.g., a human cell. In anotherembodiment, the gene is a cytokine gene, e.g., IL-2, IFN-γ, IL-4, IL-5,TNFα, TGF-β, LT (lymphotoxin), and IL-10. In yet another embodiment, thegene is a cytokine receptor gene. In certain embodiments the agentdirectly modulates T-bet expression and/or activity. In otherembodiments, the agent indirectly modulates T-bet expression and/oractivity.

In other aspects, the invention relates to a method of modulating theexpression and/or activity of T-bet, comprising contacting a cell withan agent that modulates TGF-β-mediated signaling. In one embodiment, theTGF-β-mediated signaling can be increased and T-bet expression and/oractivity decreased. In another embodiment, the TGF-β-mediated signalingis decreased and T-bet expression and/or activity is increased Themethod can be performed in a subject that would benefit from reducedIFN-γ production by cells of the innate and/or adaptive immune system.

The invention also pertains to a method of modulating the expressionand/or activity of T-bet, comprising contacting a cell with an agentthat modulates STAT1-mediated signaling. In one embodiment,STAT1-mediated signaling is increased and T-bet expression and/oractivity is increased. In another embodiment, STAT1 mediated signalingis decreased and T-bet expression and/or activity is decreased. Themethod can be performed in a subject that would benefit from increasedIFN-γ production by cells of the innate and/or adaptive immune system.

The invention further pertains to a method of modulating the interactionbetween T-bet and a molecule with which T bet interacts comprisingcontacting a cell with an agent that modulates T-bet expression and/oractivity such that the interaction between T-bet and a Molecule withwhich T-bet interacts is modulated. The molecule with which T-betinteracts can be selected from the group consisting of: a T-betresponsive DNA element, a cytokine promoter, and a Tec kinase. Themethod can be performed in a subject that would benefit from modulationof IFN-γ production by cells of the innate and/or adaptive immunesystem.

In yet another aspect, the invention pertains to a method for reducingthe level of expression of genes which are activated by extracellularinfluences which induce T-bet mediated intracellular signaling in acell, the method comprising contacting a cell with an agent that reducesT-bet activity in the cell such that expression of the genes is reduced.The cell may be a T cell, e.g., a lamina propria T cell, a CD8+ cell,and a CD4+ cell, a B cell, a dendritic cell, or NK cell. In oneembodiment the cell is a mammalian cell, e.g., a human cell. In anotherembodiment, the gene is a cytokine gene, e.g., IL-2, IFN-γ, IL-4, IL-5,TNFα, TGF-β, LT (lymphotoxin), and IL-10. In yet another embodiment, thegene is a cytokine gene, e.g., IFN-γ.

In another aspect, the invention relates to a method for increasing thelevel of expression of genes which are activated by extracellularinfluences which induce T-bet mediated intracellular signaling in acell, the method comprising contacting a cell with an agent thatincreases T-bet activity in the cell such that expression of the genesis increased. The cell may be a T cell, e.g., a lamina propria T cell, aCD8+ cell, and a CD4+ cell, a B cell, a dendritic cell, or NK cell. Inone embodiment the cell is a mammalian cell, e.g., a human cell. Inanother embodiment, the gene is a cytokine gene, e.g., IL-2, IFN-γ,IL-4, IL-5, TNFα, TGF-β, LT (lymphotoxin), and IL-10. In yet anotherembodiment, the gene is a cytokine gene, e.g., IFN-γ.

The invention also pertains to a method for modulating T-bet expressionand/or activity in a cell comprising contacting the cell with an agentthat modulates T-bet expression and/or activity such that T-betexpression and/or activity in the cell is modulated. The agent canincrease or decrease the activity of T-bet or increase or decrease theexpression of T-bet.

The invention also pertains to a method for modulating the production ofat least one cytokine by a cell comprising contacting the cell with anagent that modulates T-bet expression and/or activity such that cytokineproduction in the cell is modulated. The cell may be selected from thegroup consisting of: a T cell, a B cell, an NK cell, and a dendriticcell. The at least one cytokine can be a Th1 cytokine, e.g., The methodIFN-γ. The at least one cytokine may also be a Th2 cytokine. In oneembodiment, the method results in altered pattern of cytokines producedby the cell.

The invention further pertains to a method of modulating an immuneresponse in a subject comprising administering an agent that modulatesT-bet to the subject such that the immune response is modulated. Immuneresponses that can be modulated include, a humoral immune response and acellular immune response.

In another aspect, the invention pertains to a method of modulatingIFN-γ production by a cell comprising contacting a cell with an agentthat modulates T-bet expression and/or activity such that IFN-γproduction is modulated. The cell may be a T cell, e.g., a laminapropria T cell, a CD8+ cell, and a CD4+ cell, a B cell, a dendriticcell, or NK cell. In one embodiment the cell is a mammalian cell, e.g.,a human cell. In certain embodiment, the expression and/or activity ofT-bet is increased, thereby increasing the production of IFN-γ. In otherembodiments, the expression and/or activity of T-bet is decreased,thereby decreasing the production of IFN-γ.

The invention further pertains to a method of modulating signaling viathe Jak1/STAT-1 pathway in a cell comprising contacting a cell with anagent that modulates T-bet expression and/or activity such thatsignaling via the Jak1/STAT-1 pathway is modulated. In another aspect,the invention pertains to a method of modulating the IgG class switchingin a cell comprising contacting a cell with an agent that modulatesT-bet expression and/or activity such IgG Class Switching is modulated.In yet another aspect, the invention pertains to a method of modulatingB lymphocyte function comprising contacting a cell with an agent thatmodulates T-bet expression and/or activity such B lymphocyte function ismodulated.

In a further aspect, the invention pertains to a method of modulatingTGF-β-mediated signaling in a cell comprising contacting a cell with anagent that modulates the activity or expression of T-bet such thatTGF-β-mediated signaling is modulated. In one embodiment, the activityor expression of T-bet is decreased in the cell and TGF-β-mediatedsignaling is increased. In another embodiment, the activity orexpression of T-bet is increased in the cell and TGF-β-mediatedsignaling is decreased.

The invention also pertains to a method of modulating the generation ofCD8+ effector memory cells comprising contacting a CD8+ T cell with anagent that modulates the expression and/or activity of T-bet to therebymodulate the production of CD8+ effector memory cells. In oneembodiment, the expression and/or activity of T-bet is increased,thereby increasing the generation of CD8 effector memory cells. Inanother embodiment, the expression and/or activity of T-bet isdecreased, thereby decreasing the generation of CD8 effector memorycells. The invention further pertains to a method of treating a disorderthat would benefit from modulation of a CD8+ T cell response comprisingcontacting a CD8+ T cell with an agent that modulates the expressionand/or activity of T-bet to thereby treat a disorder that would benefitfrom modulation of a CD8+ T cell response. In one embodiment, theexpression and/or activity of T-bet is increased, thereby increasing theactivity of CD8 cells. In another embodiment, the expression and/oractivity of T-bet is decreased, thereby decreasing the activity of CD8+cells. The disorder to be treated may be a viral infection, e.g.,cancer. The invention further pertains to a method of modulating theproduction of IL-10 in a CD8+ cell comprising contacting a CD8+ T cellwith an agent that modulates the expression and/or activity of T-bet tothereby modulate the production of IL-10 in a CD8+ T cell. In certainembodiments, the expression and/or activity of T-bet is increased,thereby decreasing the production of IL-10. In other embodiments, theexpression and/or activity of T-bet is decreased, thereby increasing theproduction of IL-10.

In another aspect, the invention pertains to a method of modulating thecytolytic activity of a CD8+ cell comprising contacting a CD8+ T cellwith an agent that modulates the expression and/or activity of T-bet tothereby modulate the cytolytic activity of a CD8+ cell. The inventionalso pertains to a method of modulating the production of IFN-γ in an NKcell comprising contacting an NK cell with an agent that modulates theexpression and/or activity of T-bet to thereby modulate the productionof IFN-γ in an NK cell. In one embodiment, the expression and/oractivity of T-bet is increased, thereby increasing the production ofIFN-γ. In another embodiment, the expression and/or activity of T-bet isdecreased, thereby decreasing the production of IFN-γ.

The invention also pertains to a method of modulating the generation ofNK cells comprising contacting an NK cell with an agent that modulatesthe expression and/or activity of T-bet to thereby modulate thecytolytic activity of an NK cell. In one embodiment, the expressionand/or activity of T-bet is increased, thereby increasing the generationof NK cells. In another embodiment, the expression and/or activity ofT-bet is decreased, thereby decreasing the generation of NK cells. Theinvention also pertains to a method of modulating NK cell cytolyticactivity comprising contacting an NK cell with an agent that modulatesthe expression and/or activity of T-bet to thereby modulate NK cellcytolytic activity. In one embodiment, the agent increases theexpression and/or activity of T-bet and NK cell cytolytic activity isincreased. In another embodiment, the agent decreases the expressionand/or activity of T-bet and NK cell activity is decreased. In a certainembodiment, the expression of granzyme b is decreased. In anotherembodiment, the agent decreases the expression of perforin

In another aspect, the invention pertains to a method of modulating theproduction of IFN-γ in a dendritic cell comprising contacting an NK cellwith an agent that modulates the expression and/or activity of T-bet tothereby modulate the production of IFN-γ in a dendritic cell. In certainembodiments, the expression and/or activity of T-bet is increased,thereby increasing the production of IFN-γ. In other embodiments, theexpression and/or activity of T-bet is decreased, thereby decreasing theproduction of IFN-γ.

The invention also pertains to a method of modulating the production ofIFN-γ at the site of antigen presentation to a naive T cell in vivo,comprising administering an agent that modulates the expression and/oractivity of T-bet to a subject to thereby modulate the production ofIFN-γ at the site of antigen presentation to a naive T cell. In oneembodiment, the method further comprises administering an antigen to thesubject.

The invention also pertains to a method of modulating the induction oftolerance in peripheral T cells, comprising administering an agent thatmodulates the expression and/or activity of T-bet to a subject tothereby modulate the induction of tolerance in peripheral T cells. Inone embodiment, the number or percentage of Tr cells in the subject ismodulated. In another embodiment, the activity or expression of T-bet isincreased and peripheral tolerance is increased. In yet otherembodiments, the activity or expression of T-bet is decreased andperipheral tolerance is decreased.

In yet another aspect, the invention pertains to a method of treating orpreventing a disorder that would benefit from treatment with an agentthat modulates the expression and/or activity of T-bet comprisingadministering to a subject with said disorder an agent that modulatesthe expression and/or activity of T-bet. The invention also pertains toa method of treating or preventing a disorder that would benefit fromtreatment with an agent that modulates the expression of T-betcomprising administering to a subject with said disorder an agent thatmodulates the expression of T-bet. The disorders of the inventioninclude autoimmune diseases as described herein and known in the art.The autoimmune diseases can be mediated by Th2 cells. In particularembodiments, the autoimmunce diseases are Crohn's disease, multiplesclerosis, ulcerative colitis or rheumatod arthritis. In one embodiment,a disorder that can be treated is asthma.

The invention also pertains to a method of treating or preventing aninfectious disease that would benefit from treatment with an agent thatmodulates the expression and/or activity of T-bet comprisingadministering to a subject with said infectious disease an agent thatmodulates the expression and/or activity of T-bet. The infectiousdisease may be caused by a bacterium or by a virus.

The invention further pertains to a method of treating multiplesclerosis comprising administering an agent that reduces the expressionand/or activity of T-bet to a subject such that multiple sclerosis istreated and a method of treating rheumatoid arthritis comprisingadministering an agent that reduces the expression and/or activity ofT-bet to a subject such that rheumatoid arthritis is treated.

The invention also pertains to a method for identifying a compound thatmodulates the expression and/or activity of a T-bet polypeptide,comprising providing an indicator composition that comprises a T-betpolypeptide; contacting the indicator composition with a test compound;and determining the effect of the test compound on the expression and/oractivity of the T-bet polypeptide in the indicator composition tothereby identify a compound that modulates the expression and/oractivity of a T-bet polypeptide. The indicator composition may comprisea T-bet polypeptide and a DNA molecule to which the T-bet polypeptidebinds; and the effect of the test compound on the expression and/oractivity of the T-bet polypeptide is determined by evaluating thebinding of the T-bet polypeptide to the DNA molecule in the presence andabsence of the test compound. The indicator composition may also be acell comprising a T-bet polypeptide and a reporter gene responsive tothe T-bet polypeptide; and the effect of the test compound on theexpression and/or activity of the T-bet polypeptide is determined byevaluating the expression of the reporter gene in the presence andabsence of the test compound. The method may further comprisedetermining the effect of the test compound on an immune response tothereby identify a compound that modulates an immune response. In oneembodiment, the expression and/or activity of T-bet is enhanced. Onanother embodiment, the expression and/or activity of T-bet isinhibited.

The activity of T-bet may be IFN-γ production or transcription of IgG2a.The test compound may be selected from the group comprised of: a T-betnucleic acid molecule, a T-bet peptide, a small molecule T-bet agonistand a small molecule T-bet antagonist; an intracellular antibody, anucleic acid molecule that is antisense to a T-bet molecule, a dominantnegative T-bet molecule, a small molecule T-bet agonist and a smallmolecule T-bet antagonist. The cell may be selected from the groupconsisting of: a T cell, a B cell, and a macrophage. In one embodiment,the indicator composition is a cell that expresses T-bet polypeptide. Inanother embodiment, the cell has been engineered to express the T-betpolypeptide by introducing into the cell an expression vector encodingthe T-bet polypeptide. In another embodiment, the indicator compositionis a cell free composition. In yet another embodiment, the indicatorcomposition is a cell that expresses a T-bet polypeptide and a targetmolecule, and the ability of the test compound to modulate theinteraction of the T-bet polypeptide with a target molecule ismonitored.

In another aspect, the invention pertains to a method of identifying acompound that modulates a signal transduction pathway involving T-betcomprising: a) contacting cells deficient in T-bet with a test compound;and b) determining the effect of the test compound on an expressionand/or activity of T-bet, the test compound being identified as amodulator of the expression and/or activity of a signal transductionpathway involving T-bet based on the ability of the test compound tomodulate a signal transduction pathway involving T-bet in the cellsdeficient in T-bet. In one embodiment, the cells deficient in T-bet arein a non-human T-bet deficient animal and the cells are contacted withthe test compound by administering the test compound to the non-humanT-bet deficient animal.

The invention also relates to a method of identifying compounds usefulin modulating TGF-β-mediated signaling comprising, a) providing anindicator composition comprising T-bet polypeptide; b) contacting theindicator composition with each member of a library of test compounds;c) selecting from the library of test compounds a compound of interestthat modulates the expression and/or activity of T-bet polypeptide tothereby identify a compound that modulates TGF-β-mediated signaling.T-bet activity is measured by measuring cytokine production, measuringthe expression of Smad7, by measuring TGF-β-mediated signaling, whereinthe amount of TGF-β is measured.

The invention also relates to a method of identifying compounds usefulin modulating the Jak1/STAT-1 pathway comprising, a) providing anindicator composition comprising T-bet polypeptide; b) contacting theindicator composition with each member of a library of test compounds;c) selecting from the library of test compounds a compound of interestthat modulates the expression and/or activity of T-bet polypeptide tothereby identify a compound that modulates the Jak1/STAT-1 pathway.

The invention also encompasses a method of identifying compounds usefulin modulating T cell lineage commitment comprising, a) providing anindicator composition comprising T-bet polypeptide; b) contacting theindicator composition with each member of a library of test compounds;c) selecting from the library of test compounds a compound of interestthat modulates the activity or expression of T-bet polypeptide tothereby identify a compound that modulates T cell lineage commitment. Inone embodiment, commitment of T cells to Th1 lineage is measured. Inanother embodiment, commitment of T cells to Th2 lineage is measured. Inyet another embodiment, the compound increases commitment of T cells toTh1 lineage. In another embodiment, the compound decreases commitment ofT cells to Th2 lineage. In another embodiment, the compound increasescommitment of T cells to Th2 lineage. In yet another embodiment, thecompound decreases commitment of T cells to Th1 lineage. In certainembodiments, the activity of T-bet is measured by measuring cytokineproduction, e.g., a cytokine selected from the group consisting of IL-2,IFNγ, IL-4, IL-5, TNFα, TGF-β, LT (lymphotoxin), and IL-10. Theindicator composition can be a cell that expresses T-bet polypeptide. Inone embodiment, the cell is committed to a T cell lineage. In anotherembodiment, the cell is not yet committed to a T cell lineage.

In another aspect, the invention relates to a method of identifyingcompounds useful in modulating the production of at least one cytokinecomprising, a) providing an indicator composition comprising T-betpolypeptide; b) contacting the indicator composition with each member ofa library of test compounds; c) selecting from the library of testcompounds a compound of interest that modulates the expression and/oractivity of T-bet polypeptide to thereby identify a compound thatmodulates the production of at least one cytokine. In one embodiment,the production of at least one cytokine is measured. In anotherembodiment, the production of more than one cytokine is measured. Inanother embodiment, the pattern of cytokines produced is measured. Thecell may be selected from the group consisting of: a T cell, a B cell,and an NK cell.

The invention also relates to a method of identifying compounds usefulin modulating the production of IFN-γ comprising, a) providing anindicator composition comprising T-bet polypeptide; b) contacting theindicator composition with each member of a library of test compounds;c) selecting from the library of test compounds a compound of interestthat modulates the expression and/or activity of T-bet polypeptide tothereby identify a compound that modulates the production of IFN-γ. Theindicator composition can be a cell that expresses T-bet polypeptide.The cell can be selected from the group consisting of: a T cell, a Bcell, and an NK cell. The cell can be engineered to express the T-betpolypeptide by introducing into the cell an expression vector encodingthe T-bet polypeptide. The indicator composition can be a cell freecomposition. The indicator composition can be a cell that expresses aT-bet polypeptide and a target molecule, and the ability of the testcompound to modulate the interaction of the T-bet polypeptide with atarget molecule is monitored. In one embodiment, the indicatorcomposition comprises an indicator cell, wherein the indicator cellcomprises a T-bet polypeptide and a reporter gene responsive to theT-bet polypeptide. In another embodiment, the indicator cell contains: arecombinant expression vector encoding the T-bet polypeptide; and avector comprising a T-bet responsive regulatory element operativelylinked a reporter gene; and said method comprises: a) contacting theindicator cell with a test compound; b) determining the level ofexpression of the reporter gene in the indicator cell in the presence ofthe test compound; and c) comparing the level of expression of thereporter gene in the indicator cell in the presence of the test compoundwith the level of expression of the reporter gene in the indicator cellin the absence of the test compound to thereby select a compound ofinterest that modulates the expression and/or activity of T-betpolypeptide.

The invention also relates to a method of identifying a compound thatmodulates the production of IFN-γ comprising: a) contacting cellsdeficient in T-bet with a test compound; and b) determining the effectof the test compound on the production of IFN-γ the test compound beingidentified as a modulator of the production of IFN-γ based on theability of the test compound to modulate the production of IFN-γ in thecells deficient in T-bet. The invention also relates to a method ofidentifying compounds useful in modulating IFN-γ production comprising,a) providing an indicator composition comprising T-bet polypeptide; b)contacting the indicator composition with each member of a library oftest compounds; c) selecting from the library of test compounds acompound of interest that modulates the interaction between T-bet and aTec kinase to thereby identify compounds useful in modulating IFN-γproduction.

The invention further relates to a method of identifying compoundsuseful in modulating IL-4 production comprising, a) providing anindicator composition comprising T-bet polypeptide; b) contacting theindicator composition with each member of a library of test compounds;c) selecting from the library of test compounds a compound of interestthat modulates the interaction between T-bet and a Tec kinase to therebyidentify compounds useful in modulating IL-4 production.

The invention further relates to a method of identifying compoundsuseful in modulating IgG class switching comprising, a) providing anindicator composition comprising T-bet polypeptide; b) contacting theindicator composition with each member of a library of test compounds;c) selecting from the library of test compounds a compound of interestthat modulates the expression and/or activity of T-bet polypeptide; andd) determining the effect of the compound of interest on IgG classswitching to thereby identify a compound that modulates the productionof IgG class switching. In one embodiment, the activity of T-bet ismeasured by measuring IgG2a production. The T-bet activity can also bemeasured by measuring IgG1 production or by measuring IgE production.

In another aspect, the invention relates to a method of identifyingcompounds useful in modulating B lymphocyte function comprising, a)providing an indicator composition comprising T-bet polypeptide; b)contacting the indicator composition with each member of a library oftest compounds; c) selecting from the library of test compounds acompound of interest that modulates the expression and/or activity ofT-bet polypeptide; and d) determining the effect of the compound ofinterest on B lymphocyte function to thereby identify a compound thatmodulates B lymphocyte function. The activity of T-bet is measured bymeasuring, for example, cytokine production or the production ofpathogenic autoantibodies

In yet another aspect, the invention pertains to a method of identifyingcompounds useful in modulating an autoimmune disease comprising, a)providing an indicator composition comprising T-bet polypeptide; b)contacting the indicator composition with each member of a library oftest compounds; c) selecting from the library of test compounds acompound of interest that modulates the expression and/or activity ofT-bet polypeptide to thereby identify a compound that modulates anautoimmune disease.

The invention further pertains to a method for modulating the expressionand/or activity of T-bet comprising contacting a cell with an agent thatmodulates a post-translational modification of T-bet such that theexpression and/or activity of T-bet is modulated. The post-translationalmodification is selected from, for example, phosphorylation,glycosylation and ubiquitination.

In another aspect, the invention pertains to a method of diagnosing asubject for a disorder associated with aberrant immune cell activationcomprising: detecting expression of T-bet in immune cells of a subjectsuspected of having said disorder; comparing expression of T-bet inimmune cells of said subject to a control that is not associated withaberrant immune cell activation; and diagnosing the subject for adisorder based on a change in expression of T-bet in immune cells of thesubject as compared to the control. The disorder can be, for example, anautoimmune disease as described herein or known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A) an amino acid sequence alignment of human T-bet (SEQ IDNO:2) and murine T-bet (SEQ ID NO:4), and (B) a nucleotide sequencealignment of human T-bet (SEQ ID NO:1) and murine T-bet (SEQ ID NO:3).

FIGS. 2A-B show that T-bet binds to and transactivates consensus T-boxsites with functionally important domains that map to both 5′ and 3′regions.

FIG. 3A shows that T-bet is preferentially expressed in double negativethymocytes. Panel B shows that in a survey of Th clones, T-betexpression is restricted to Th1 cells. Panel C shows western blotanalysis of T-bet. Panel D shows FACS analysis of T-bet expression.

FIGS. 4A-B show that T-bet expression correlates with IFN-γ induction inNK and B cells.

FIG. 5 shows that T-bet transactivates the IFN-γ gene in Th cells.

FIG. 6 shows that retroviral gene transduction of T-bet reducesincreases IFN-γ production and represses IL-2 production.

FIG. 7 shows that T-bet activates IFN-γ and represses IL-2 production inprimary T cells.

FIG. 8 shows that T-bet induces IFN-γ and inhibits IL-4 production indeveloping Th2 cells.

FIG. 9 shows that T-bet redirects polarized Th2 cells into the Th1pathway. Th-skewing was carried out as above and retroviral infectionswere performed on day 9 of culture

FIG. 10 shows that T-bet redirects polarized Tc2 cells into the Tc1pathway. CD8+ T cells were purified by MoFlo and cultured under Th2skewing conditions as above and retroviral transductions performed onday 8 of culture.

FIG. 11 shows that T-bet is tyrosine phosphorylated.

FIG. 12 shows the activity of a T-bet dominant negative mutant.

FIG. 13 shows that treatment of activated T-cell enriched LPMCs withTGF-β but not IL-4 suppressed T-bet expression suggesting a reciprocalrelationship between TGF-β and T-bet.

FIG. 14 shows that mice lacking T-bet are resistant to EAE development.

FIG. 15 shows that mice lacking T-bet had a mean clinical score of lessthan 0.5 and were protected from EAE.

FIG. 16 shows that the percentage of CD4⁺ cells staining positive forIFN-γ was 33% in 2D2 MOG×T-bet^(+/+) animals and 3% in 2D2MOG×T-bet^(−/−) animals.

FIG. 17 shows a substantial decrease (approximately two thirdsreduction) in numbers of CD8 cells in the absence of T-bet as evidencedby the reduction in CD8⁺, CD44Hi, CD62Lhi, CD69Hi and Ly6CHi cells.

FIG. 18 shows that T-bet is required for IFN-γ production in CD8+ cells.

FIG. 19 shows that T-bet regulates the production of IFN-γ in NK cells.

FIG. 20 shows that the generation of NK cells is also impaired in theabsence of T-bet.

FIG. 21 shows diminished spontaneous tumor cell lysis in T-bet^(−/−) NKcells.

FIG. 22 shows that expression of lytic genes was impaired in the absenceof T-bet.

FIG. 23 A-C show the normal development and activation of murinedendritic cells in mice lacking T-bet.

FIG. 24 shows low levels of T-bet expression in unstimulated DCs and arapid up-regulation of T-bet transcript levels after treatment withIFN-γ.

FIG. 25 shows that T-bet is essential for optimal production of IFN-γ bydendritic cells.

FIG. 26 shows the conserved structure of Tec family members.

FIG. 27 shows the predicted tyrosine phosphorylation sites of humanT-bet.

FIG. 28 shows the modified forms of T-bet that were made and used assubstrates in in vitro kinase assays.

FIG. 29 shows that both ITK and Rlk phosphorylated N-terminal andC-terminal but not DNA-binding regions of T-bet in in vitro kinaseassays.

FIG. 30 shows that although T-bet is present in T cells from ITK knockout animals, tyrosine phosphorylation of the molecule is reduced. Incontrast, T-bet was hyperphosphorylated in Rlk knockout T cells

FIG. 31 shows that T_(R) cells are increased in the absence of T-bet.

FIG. 32 shows that IFN-γ regulates T-bet expression in aself-reinforcing feedback mechanism between IFN-γ and T-bet.

FIG. 33 shows that T-bet expression was markedly reduced in STAT1 andIFN-γ R1 deficient T cells yet STAT4 deficient cells exhibited T-betlevels comparable to wildtype controls.

FIG. 34 shows reduced IFN-γ expression in T-bet and STAT1, but not STAT4deficient CD4+ T cells.

FIG. 35A-B shows reduced IFN-γ expression in T-bet, STAT1 and STAT4deficient Th1 cells.

FIG. 36 shows that CD8 cells that lack T-bet produce substantiallyincreased levels of IL-10 and IL-2.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to T-bet compositions, such as isolated nucleicacid molecules encoding T-bet and isolated T-bet proteins, as well asmethods of use therefore. The expression and/or activity of T-bet can bemodulated using the instant invention. As discussed in more detailbelow, T-bet is an important intracellular transducer or mediator of avariety of extracellular signals. T-bet is a transcription factor thatoperates in different cell types to transduce extracellular signals intospecific patterns of gene expression. In particular, it has now beendemonstrated that T-bet has a central role in cytokine gene expression.Different cell types and different genes respond to T-bet, which servesto modulate a variety of cellular responses. T-bet also controlsexpression of several genes, expression of these genes and otherssimilarly affected can be modulated (e.g., enhanced or reduced) bycontrolling the expression and/or activity of T-bet.

Using the instant invention, the expression of a gene responsive toT-bet can be positively or negatively regulated to provide for increasedor decreased production, respectively, of the protein whose expressionis modulated by T-bet. Furthermore, genes which do not, in theirnaturally-occurring form, have T-bet recognition sequences can be placedunder the control of T-bet by inserting a T-bet binding site in anappropriate position using techniques known in the art. In addition,extracellular signals transduced via T-bet can be modulated, e.g., bycontacting a cell with an agent that modulates the expression and/oractivity of T-bet such that the T-bet mediated effects of suchextracellular influences are modulated. Thus, T-bet expression and/oractivity can be controlled to modulate signaling via a signaltransduction pathway in which T-bet is involved.

So that the invention may be more readily understood, certain terms arefirst defined.

As used herein, the term “modulated” with respect to T-bet includeschanging the expression, activity or function of T-bet in such a mannerthat it differs from the naturally-occurring expression, function oractivity of T-bet under the same conditions.

For example, the expression, function or activity can be greater or lessthan that of naturally occurring T-bet, e.g., owing to a change inbinding specificity, etc. As used herein, the various forms of the term“modulate” include stimulation (e.g., increasing or upregulating aparticular response or activity) and inhibition (e.g., decreasing ordownregulating a particular response or activity).

As used herein, the term “T-bet molecules” includes T-bet nucleic acidmolecules that share structural features with the nucleic acid moleculesshown in SEQ ID NOs: 1 and 3 and T-bet proteins that share thedistinguishing structural and functional features of the T-bet proteinsshown in SEQ ID NOs 2 and 4. The T-bet proteins are members of the T-boxfamily of proteins and share some amino acid sequence homology toBrachyury, Tbx1-6, T-brain-1 (Tbr-1). T-box proteins comprise a T boxdomain which binds to DNA at a T box binding site. Further structuraland functional features of T-bet proteins are provided below.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g.,mRNA). The nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An used herein, an “isolated nucleic acid molecule” refers to a nucleicacid molecule that is free of gene sequences which naturally flank thenucleic acid in the genomic DNA of the organism from which the nucleicacid is derived (i.e., genetic sequences that are located adjacent tothe gene for the isolated nucleic molecule in the genomic DNA of theorganism from which the nucleic acid is derived). For example, invarious embodiments, an isolated T-bet nucleic acid molecule typicallycontains less than about 10 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived, and more preferably contains less thanabout 5, kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of naturallyflanking nucleotide sequences. An “isolated” T-bet nucleic acid moleculemay, however, be linked to other nucleotide sequences that do notnormally flank the T-bet sequences in genomic DNA (e.g., the T-betnucleotide sequences may be linked to vector sequences). In certainpreferred embodiments, an “isolated” nucleic acid molecule, such as acDNA molecule, also may be free of other cellular material. However, itis not necessary for the T-bet nucleic acid molecule to be free of othercellular material to be considered “isolated” (e.g., a T-bet DNAmolecule separated from other mammalian DNA and inserted into abacterial cell would still be considered to be “isolated”).

As used herein, the term “hybridizes under high stringency conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences having substantial homology (e.g., typicallygreater than 70% homology) to each other remain stably hybridized toeach other. A preferred, non-limiting example of high stringencyconditions are hybridization in a hybridization buffer that contains 6×sodium chloride/sodium citrate (SSC) at a temperature of about 45° C.for several hours to overnight, followed by one or more washes in awashing buffer containing 0.2×SSC, 0.1% SDS at a temperature of about50-65° C.

The term “percent (%) identity” as used in the context of nucleotide andamino acid sequences (e.g., when one amino acid sequence is said to be X% identical to another amino acid sequence) refers to the percentage ofidentical residues shared between the two sequences, when optimallyaligned. To determine the percent identity of two nucleotide or aminoacid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps may be introduced in one sequence for optimalalignment with the other sequence). The residues at correspondingpositions are then compared and when a position in one sequence isoccupied by the same residue as the corresponding position in the othersequence, then the molecules are identical at that position. The percentidentity between two sequences, therefore, is a function of the numberof identical positions shared by two sequences (i.e., % identity=# ofidentical positions/total # of positions×100).

Computer algorithms known in the art can be used to optimally align andcompare two nucleotide or amino acid sequences to define the percentidentity between the two sequences. A preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Research25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov. For example, thenucleotide sequences of the invention were blasted using the defaultBlastn matrix 1-3 with gap penalties set at: existance 5 and extension2. The amino acid sequences of the invention were blasted using thedefault settings: the Blosum62 matrix with gap penalties set atexistance 11 and extension 1.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. If multiple programs are used tocompare sequences, the program that provides optimal alignment (i.e.,the highest percent identity between the two sequences) is used forcomparison purposes.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, an “antisense” nucleic acid comprises a nucleotidesequence which is complementary to a “sense” nucleic acid encoding aprotein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, complementary to an mRNA sequence or complementary to thecoding strand of a gene. Accordingly, an antisense nucleic acid canhydrogen bond to a sense nucleic acid.

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”or simply “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid of the invention, such as a recombinant expressionvector of the invention, has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It should beunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

As used herein, a “transgenic animal” refers to a non-human animal,preferably a mammal, more preferably a mouse, in which one or more ofthe cells of the animal includes a “transgene”. The term “transgene”refers to exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, for example directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal.

As used herein, a “homologous recombinant animal” refers to a type oftransgenic non-human animal, preferably a mammal, more preferably amouse, in which an endogenous gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material and culturemedium when isolated from cells or produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the term “antibody” is intended to includeimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as Fab and F(ab′)₂ fragments. The terms “monoclonal antibodies” and“monoclonal antibody composition”, as used herein, refer to a populationof antibody molecules that contain only one species of an antigenbinding site capable of immunoreacting with a particular epitope of anantigen, whereas the term “polyclonal antibodies” and “polyclonalantibody composition” refer to a population of antibody molecules thatcontain multiple species of antigen binding sites capable of interactingwith a particular antigen. A monoclonal antibody compositions thustypically display a single binding affinity for a particular antigenwith which it immunoreacts.

As used here, the term “intrabodies” refers to intracellularly expressedantibody constructs, usually single-chain Fv (scFv) antibodies, directedagainst a target inside a cell, e.g. an intracellular protein such asT-bet.

As used herein, the term “autoimmune disease” refers to disorders orconditions in a subject wherein the immune system attacks the body's owncells, causing tissue destruction. Autoimmune diseases include generalautoimmune diseases, i.e., in which the autoimmune reaction takes placesimultaneously in a number of tissues, or organ specific autoimmunediseases, i.e., in which the autoimmune reaction targets a single organ.Examples of autoimmune diseases that can be diagnosed, prevented ortreated by the methods and compositions of the present inventioninclude, but are not limited to, diabetes mellitus, rheumatoidarthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriaticarthritis, multiple sclerosis, myasthenia gravis, systemic lupuserythematosis, autoimmune thyroiditis, atopic dermatitis and eczematousdermatitis, psoriasis, Sjögren's Syndrome, alopecia areata, allergicresponses due to arthropod bite reactions, Crohn's disease, aphthousulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, compound eruptions, leprosy reversal reactions, erythemanodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegner's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, experimental allergicencephalomyelitis (EAE), and interstitial lung fibrosis, and secondarydiseases caused as a result of autoimmune diseases.

As used herein, the term “T cell” (i.e., T lymphocyte) is intended toinclude all cells within the T cell lineage, including thymocytes,immature T cells, mature T cells and the like, from a mammal (e.g.,human or mouse).

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune. Exemplary immune responses include T cellresponses, e.g., cytokine production, and cellular cytotoxicity. Inaddition, the term immune response includes antibody production (humoralresponses) and activation of cells of the innate immune system, e.g.,cytokine responsive cells such as macrophages.

As used herein, the term “T helper type 1 response” refers to a responsethat is characterized by the production of one or more cytokinesselected from IFN-γ, IL-2, TNF, and lymphotoxin (LT) and other cytokinesproduced preferentially or exclusively by Th1 cells rather than by Th2cells.

As used herein, a “T helper type 2 response” (Th2 response) refers to aresponse by CD4⁺ T cells that is characterized by the production of oneor more cytokines selected from IL-4, IL-5, IL-6 and IL-10, and that isassociated with efficient B cell “help” provided by the Th2 cells (e.g.,enhanced IgG1 and/or IgE production).

As used herein, the term “a cytokine that regulates development of a Th2response” is intended to include cytokines that have an effect on theinitiation and/or progression of a Th2 response, in particular,cytokines that promote the development of a Th2 response, e.g., IL-4,IL-5 and IL-10.

As used herein the term “innate immune system” includes natural ornative immune mechanisms, i.e., mechanisms that exist before infection,are capable of rapid responses to microbes, and react in essentially thesame way to repeated infections.

As used herein, the term “adaptive immune system” or “specific immunesystem” includes immune mechanisms that are stimulated by exposure ofinfectious agents and increase in magnitude and defensive capabilitieswith each successive exposure to a particular microbe.

The term “agent” or “compound” or “test compound” includes reagents ortest agents which are employed in the methods or assays or present inthe compositions of the invention. The term “agent” or “compound” or“test compound” includes compounds that have not previously beenidentified as, or recognized to be, a modulator of T-bet expression oractivity. In one embodiment, more than one compound, e.g., a pluralityof compounds, can be tested at the same time in a screening assay fortheir ability to modulate expression and/or activity of T-bet or amolecule acting upstream or downstream of T-bet in a signal transductionpathway. The term “library of test compounds” refers to a panelcomprising a multiplicity of test compounds.

In one embodiment, the term “agent” or “compound” or “test compound”excludes naturally occurring compounds such as cytokines. In anotherembodiment, the term agent excludes antibodies which bind to naturallyoccurring cytokines. In another embodiment, the term “agent” excludesantibodies that bind to cytokine receptors. In yet another embodiment,the term “agent” excludes those agents that transduce signals via the Tcell receptor, e.g., antigen in the context of an MHC molecule orantibody to a component of the T cell receptor complex. In oneembodiment, the agent or test compound is a compound that directlyinteracts with T-bet or directly interacts with a molecule with whichT-bet interacts (e.g., a compound that inhibits or stimulates theinteraction between T-bet and a T-bet target molecule, e.g., DNA oranother protein). In another embodiment, the compound is one thatindirectly modulates T-bet expression and/or activity, e.g., bymodulating the activity of a molecule that is upstream or downstream ofT-bet in a signal transduction pathway involving T-bet. Such compoundscan be identified using screening assays that select for such compounds,as described in detail below.

The term “small molecule” is a term of the art and includes moleculesthat are less than about 1000 molecular weight or less than about 500molecular weight. In one embodiment, small molecules do not exclusivelycomprise peptide bonds. In another embodiment, small molecules are notoligomeric. Exemplary small molecule compounds which can be screened foractivity include, but are not limited to, peptides, peptidomimetics,nucleic acids, carbohydrates, small organic molecules (e.g.,polyketides) (Cane et al. 1998. Science 282:63), and natural productextract libraries. In another embodiment, the compounds are small,organic non-peptidic compounds. In a further embodiment, a smallmolecule is not biosynthetic.

As used herein, the term “a modulator of T-bet” includes a modulator ofT-bet expression, processing, post-translational modification, oractivity. The term includes agents, for example a compound or compoundswhich modulates transcription of a T-bet gene, processing of a T-betmRNA, translation of T-bet mRNA, post-translational modification of aT-bet protein (e.g., glycosylation, ubiquitinization or phosphorylation)or activity of a T-bet protein. A “modulator of T-bet activity” includescompounds that directly or indirectly modulate T-bet activity. Forexample, an indirect modulator of T-bet activity may modulate a signaltransduction pathway that includes T-bet. Examples of modulators thatdirectly modulate T-bet activity include antisense nucleic acidmolecules that bind to T-bet mRNA or genomic DNA, intracellularantibodies that bind to T-bet intracellularly and modulate (i.e.,inhibit) T-bet activity, T-bet peptides that inhibit the interaction ofT-bet with a target molecule and expression vectors encoding T-bet thatallow for increased expression of T-bet activity in a cell, dominantnegative forms of T-bet, as well as chemical compounds that act tospecifically modulate the activity of T-bet.

As used herein an “agonist” of the T-bet proteins can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of a T-bet protein. An “antagonist” of a T-betprotein can inhibit one or more of the activities of the naturallyoccurring form of the T-bet protein by, for example, competitivelymodulating a cellular activity of a T-bet protein.

As used interchangeably herein, “T-bet activity,” “biological activityof T-bet” or “functional activity T-bet,” include an activity exerted byT-bet protein on a T-bet responsive cell or tissue, e.g., a T cell,dendritic cells, NK cells, or on a T-bet target molecule, e.g., anucleic acid molecule or protein target molecule, as determined in vivo,or in vitro, according to standard techniques. In one embodiment, T-betactivity is a direct activity, such as an association with aT-bet-target molecule. Alternatively, a T-bet activity is an indirectactivity, such as a downstream biological event mediated by interactionof the T-bet protein with a T-bet target molecule. The biologicalactivities of T-bet are described herein and include, but are notlimited to: modulation of IFN-γ production in cells of the innate andadaptive immune system, modulation of T cell lineage commitment,modulation of the production of cytokines, modulation of TGF-β mediatedsignaling, modulation of the Jak1/STAT-1 pathway, modulation of IgGclass switching, modulation of B lymphocyte function, and modulation ofdisorders that would benefit from modulation of T-bet, e.g., autoimmunediseases, multiple sclerosis or rheumatoid arthritis, infection, e.g.,with a virus or a bacterium, asthma, and other disorders or unwantedconditions in which Th1 or Th2 cytokines are implicated, e.g.,inflammation. These findings provide for the use of T-bet (and othermolecules in the pathways in which T-bet is involved) as drug targetsand as targets for therapeutic intervention in various diseases,disorders or conditions. The invention yet further providesimmunomodulatory compositions, such as vaccines, comprising agents whichmodulate T-bet activity.

As used herein, the term “target molecule” or “binding partner” is amolecule with which T-bet binds or interacts in nature, and whichinteraction results in a biological response. The target molecule can bea protein or a nucleic acid molecule. Exemplary target molecules of theinvention include proteins in the same signaling pathway as the T-betprotein, e.g., proteins which may function upstream (including bothstimulators and inhibitors of activity) or downstream of the T-betprotein in a pathway involving for example, modulation of T cell lineagecommitment, modulating the production of cytokines, modulating TGF-βmediated signaling, modulating the Jak1/STAT-1 pathway, modulating IgGclass switching, modulating B lymphocyte function, and modulating anautoimmune disease. Exemplary T-bet target molecules include tyrosinekinases s, e.g., a Tec kinase such as ITK or rlk or DNA sequences withwhich T-bet interacts to modulate gene transcription.

As used herein, the term “gene whose transcription is regulated byT-bet”, includes genes having a regulatory region regulated by T-bet.Such genes can be positively or negatively regulated by T-bet. The termalso includes genes which are indirectly modulated by T-bet, i.e., aremodulated as the result of the activation of a signaling pathway inwhich T-bet is involved. Exemplary genes regulated by T-bet include, forexample, the cytokine genes, e.g., IL-2, IFN-γ, IL-4, IL-5, TNFα, TGF-β,LT (lymphotoxin), and IL-10.

As used herein, the term “signal transduction pathway” includes themeans by which a cell converts an extracellular influence or signal(e.g., a signal transduced by a receptor on the surface of a cell, suchas a cytokine receptor or an antigen receptor) into a cellular response(e.g., modulation of gene transcription). Exemplary signal transductionpathways include the JAK1/STAT-1 pathway (Leonard, W. 2001. Int. J.Hematol. 73:271) and the TGF-β pathway (Attisano and Wrana. 2002.Science. 296:1646) A “signal transduction pathway involving T-bet” isone in which T-bet is a signaling molecule which relays signals.

As used herein, the term “contacting” (i.e., contacting a cell e.g. acell, with a compound) includes incubating the compound and the celltogether in vitro (e.g., adding the compound to cells in culture) aswell as administering the compound to a subject such that the compoundand cells of the subject are contacted in vivo. The term “contacting”does not include exposure of cells to a T-bet modulator that may occurnaturally in a subject (i.e., exposure that may occur as a result of anatural physiological process).

As used herein, the term “dominant negative T-bet protein” includesT-bet molecules (e.g., portions or variants thereof) that compete withnative (i.e., naturally occurring wild-type) T-bet molecules, but whichdo not have T-bet activity. Such molecules effectively decrease T-betactivity in a cell. As used herein, “dominant negative T-bet protein”refers to a modified form of T-bet which is a potent inhibitor of T-betactivity.

As used herein, the term “indicator composition” refers to a compositionthat includes a protein of interest (e.g., T-bet), for example, a cellthat naturally expresses the protein, a cell that has been engineered toexpress the protein by introducing an expression vector encoding theprotein into the cell, or a cell free composition that contains theprotein (e.g., purified naturally-occurring protein orrecombinantly-engineered protein).

As used herein, the term “cell” includes prokaryotic and eukaryoticcells. In one embodiment, a cell of the invention is a bacterial cell.In another embodiment, a cell of the invention is a fungal cell, such asa yeast cell. In another embodiment, a cell of the invention is avertebrate cell, e.g., an avian or mammalian cell. In a preferredembodiment, a cell of the invention is a murine or human cell.

As used herein, the term “dendritic cell” refers to a type ofantigen-presenting cells which are particularly active in stimulating Tcells. Dendritic cells can be obtained by culturing bone-marrow cells inthe presence of GM-CSF and selecting those cells that express MHC classII molecules and CD11c. Dendritic cells can also express CD11b⁺,DEC-205⁺, CD8-alpha⁺.

As used herein, the term “site of antigen presentation to a naïve Tcell” includes those sites within lymphoid tissues where naive CD4 Tcells first come into contact with antigen, e.g., as presented byinterdigitating dendritic cells during an in vivo primary immuneresponse.

As used herein, the term “CD8+ effector memory cell” refers to CD8 Tcells responsible for maintaining long term immunity against aparticular pathogen. CD8 effector/memory cells are CD8+, CD44 High, CD62High, CD69 High, and Ly6C High.

As used herein, the term “cytolytic activity” refers to ability of acell, e.g., a CD8+ cell or an NK cell, to lyse target cells. Suchcytolytic activity can be measured using standard techniques, e.g., byradioactively labeling the target cells.

As used herein, the term “T_(R) cell” refers to CD25 expressing CD4+ Tcells (Treg) that function as suppressors of self-specific T cellresponses. TR cells are responsible for maintenance of tolerance inperipheral T cells.

As used herein, the term “peripheral T cells” refers to mature singlepositive T cells that leave the thymus and enter the peripheralcirculation. As used herein, the term “Tec kinase” refers to a family oftyrosine kinases of which ITK and Rlk/Txk (rlk) are the predominantfamily members. Tec kinases are expressed in T cells, and are involvedin T cell antigen receptor mediated activation of T cells. The Tecfamily of protein tyrosine kinases play an important role in signalingthrough antigen-receptors such as the TCR, BCR and Fcε receptor. Membersof the Tec kinase family of tyrosine kinases include, for example, Tec,Btk, Itk, Rlk and Bmx.

As used herein, the term “tolerance” refers to unresponsiveness to Tcell-receptor-mediated signal by a T cell. Such T cell-mediated signalsinclude, e.g., antigen in the context of MHC molecules or foreign MHCmolecules.

As used herein, the term “lineage commitment” refers to the program thatinitiates T cell lineage development from a precursor cell, e.g., a Thpcell, into a fully differentiated effector cell of a specific lineage,e.g., into a T cell that secretes a specific profile of cytokines uponreceptor-mediated stimulation, such as a Th1 or a Th2 cell.

As used herein, the term “engineered” (as in an engineered cell) refersto a cell into which a nucleic acid molecule encoding the T-bet proteinhas been introduced.

As used herein, the term “cell free composition” refers to an isolatedcomposition, which does not contain intact cells. Examples of cell freecompositions include cell extracts and compositions containing isolatedproteins.

As used herein, the term “reporter gene” refers to any gene thatexpresses a detectable gene product, e.g., RNA or protein. Preferredreporter genes are those that are readily detectable. The reporter genemay also be included in a construct in the form of a fusion gene with agene that includes desired transcriptional regulatory sequences orexhibits other desirable properties. Examples of reporter genes include,but are not limited to CAT (chloramphenicol acetyl transferase) (Altonand Vapnek (1979), Nature 282: 864-869) luciferase, and other enzymedetection systems, such as beta-galactosidase; firefly luciferase (deWetet al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase(Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al.(1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al.(1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl.Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen andMalim (1992) Methods in Enzymol. 216:362-368) and green fluorescentprotein (U.S. Pat. No. 5,491,084; WO 96/23898).

As used herein, the term “T-bet-responsive element” refers to a DNAsequence that is directly or indirectly regulated by the activity ofT-bet (whereby activity of T-bet can be monitored, for example, viatranscription of the reporter genes).

As used herein, the term “cells deficient in T-bet” is intended toinclude cells of a subject that are naturally deficient in T-bet, aswells as cells of a non-human T-bet deficient animal, e.g., a mouse,that have been altered such that they are deficient in T-bet. The term“cells deficient in T-bet” is also intended to include cells isolatedfrom a non-human T-bet deficient animal or a subject that are culturedin vitro.

As used herein, the term “non-human T-bet deficient animal” refers to anon-human animal, preferably a mammal, more preferably a mouse, in whichan endogenous gene has been altered by homologous recombination betweenthe endogenous gene and an exogenous DNA molecule introduced into a cellof the animal, e.g., an embryonic cell of the animal, prior todevelopment of the animal, such that the endogenous T-bet gene isaltered, thereby leading to either no production of T-bet or productionof a mutant form of T-bet having deficient T-bet activity. Preferably,the activity of T-bet is entirely blocked, although partial inhibitionof T-bet activity in the animal is also encompassed. The term “non-humanT-bet deficient animal” is also intended to encompass chimeric animals(e.g., mice) produced using a blastocyst complementation system, such asthe RAG-2 blastocyst complementation system, in which a particular organor organs (e.g., the lymphoid organs) arise from embryonic stem (ES)cells with homozygous mutations of the T-bet gene.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid molecule and the aminoacid sequence encoded by that nucleic acid molecule, as defined by thegenetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA,ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid(Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu,E) GAA,GAG Glutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGTHistidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine(Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe,F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signalTAA, TAG, TGA (end)An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNAmolecule coding for a T-bet protein of the invention (or any portionthereof) can be use to derive the T-bet amino acid sequence, using thegenetic code to translate the DNA or RNA molecule into an amino acidsequence. Likewise, for any T-bet-amino acid sequence, correspondingnucleotide sequences that can encode the T-bet protein can be deducedfrom the genetic code (which, because of its redundancy, will producemultiple nucleic acid sequences for any given amino acid sequence).Thus, description and/or disclosure herein of a T-bet nucleotidesequence should be considered to also include description and/ordisclosure of the amino acid sequence encoded by the nucleotidesequence. Similarly, description and/or disclosure of a T-bet amino acidsequence herein should be considered to also include description and/ordisclosure of all possible nucleotide sequences that can encode theamino acid sequence.

Brachyury or T is the founding member of a family of transcriptionfactors that share a 200 amino acid DNA-binding domain called the T-box(reviewed in Smith, 1997; Papaioannou, 1997; Meisler, 1997). TheBrachyury (Greek for ‘short tail’) mutation was first described in 1927in heterozygous mutant animals who had a short, slightly kinked tail(Herrmann et al., 1990). The amino-terminal half (amino acids 1-229) ofthe Brachyury T-box protein contains a conserved domain known as the Tbox which has been shown to exhibit sequence-specific DNA-bindingactivity (Kispert, A. & Herrmann, B. G. 1993. EMBO J. 12:3211;Papapetrou, C., et al. 1997. FEBS Lett. 409:201; Kispert, A., et al.1995. EMBO J. 14:4763). The C-terminal half contains two pairs oftransactivation and repression domains. The similarity of sequencebetween the T box region in orthologous species can be as high as 99%and is around 40-70% between non-orthologous genes. The T-box domain hasrecently been co-crystallized with DNA and demonstrates a novelsequence-specific DNA recognition architecture in which the proteincontacts DNA in both the major and minor grooves (Müller, C. W. &Herrmann, B. G. 1997. Nature 389, 884).

A yeast one hybrid approach was used to identify Th-1 specifictranscription factors. Yeast cells were made to express an IL-2promoter-reporter gene construct and were transformed with a cDNAlibrary made from an anti-CD3 activated Th1 cell clone. Inspection ofthe IL-2 promoter reveals an excellent T-box binding site at −240 to−220 just 5′ of the NFkB site. As described in the appended examples,T-bet was isolated in a yeast one hybrid screening assay based on itsability to bind to the IL-2 promoter.

The nucleotide sequence encoding murine T-bet is shown in SEQ ID NO:3.Murine T-bet is a 530 amino acid protein with a 190 amino acid T-boxdomain located at residues 136-326. The amino acid sequence of murineT-bet is shown in SEQ ID NO:4. After the murine T-bet sequence wascloned as described herein, it was possible to compile the sequence ofthe human ortholog of T-bet from nucleic acid fragments which were notpreviously known to encode any known protein. The nucleotide sequence ofhuman T-bet is shown in SEQ ID NO:1. Human T-bet is a 535 amino acidprotein with a 190 amino acid T-box domain located at residues 138-327.The human T-bet gene maps to chromosome 17. The nucleotide and aminoacid sequences of two members (human and mouse) of the T-bet family ofproteins are shown in FIG. 1 and SEQ ID Nos: 1-4.

The T-bet proteins of the invention have homology to T-box proteins.There are now eight T-box genes in the mouse not including Brachyury.These include Tbx1-6, T-brain-1 (Tbr-1) and now, T-bet, each with adistinct and usually complex expression pattern. T-brain-1 expression,for example is largely restricted to distinct domains within thecerebral cortex (Bulfone, A., et al. 1995. Neuron 15, 63. T-bet is mostsimilar in sequence to Tbr-1. Outside of the T-box, the T-bet proteinsof the invention bear no similarity to other T-box proteins.

T-bet is T-box protein expressed only in T cells and is most similar insequence to Tbr-1. Other species also express Brachyury-like genes. Suchvertebrate species include Xenopus, zebrafish, chick and humans (Rao,1994; Horb and Thomsen, 1997; Conlon et al., 1996; Ryan et al., 1996;Schulte-Merker et al., 1994; Edwards et al., 1996; Morrison et al.,1996; Law et al., 1995; Cambell et al., 1998) as well as more distantspecies such as amphioxus, ascidians, echinoderms, Caenorhabditiselegans, Drosophila and other insects (Holland et al., 1995). Thesegenes are conserved both in sequence and in expression pattern.

T-bet is unique in that it is the only T-box protein to be tyrosinephosphorylated. There are three predicted tyrosine phosphorylation sitesat Tyr 76, Tyr 119, and Tyr 531 of human T-bet and one at Tyr 525 ofmurine T-bet. A nuclear localization sequence is also present at aminoacids 498-501 of human T-bet and 493-496 of murine T-bet. Mappingexperiments locate two transactivation domains, one 5′ and one 3′ of theT-box domain. The data shown herein demonstrate that T-bet binds to aconsensus T-box site (defined by target site selection in vitro as5′-GGGAATTTCACACCTAGGTGTGAAATTCCC-3′) (SEQ ID NO:5) and to the humanIL-2 promoter, the murine IL-2 promoter, the human IFN-γ intron III, andtwo binding sites in the murine IFN-γ proximal promoter. (Szabo et al.2000. Cell 100:655-669).

T-bet is expressed only in the thymus and in the peripheral lymphoidsystem. In the periphery, T-bet is expressed only in Th1 cells where itis induced both in response to TcR stimulation and to IL-12. In thethymus levels of T-bet are highest in DN and Rag2−/−thymocytes.

These data demonstrate that the selective expression of T-bet, a novelT-box family member, accounts for tissue-specific IFN-γ expression.T-bet is expressed only in Th1 and not in Th2 cells and is induced inthe former upon transmission of signals through the T cell receptor.

In addition, T-bet is a potent transactivator of the IFN-γ gene. Theexpression of T-bet correlates with IFN-γ expression in cells of theadaptive and innate immune system including: Th1 cells, B cells, NKcells, and dendritic cells. T-bet is responsible for the genetic programthat initiates Th1 lineage development from naïve Thp cells and actsboth by initiating Th1 genetic programs and by repressing the opposingprograms in Th2 cells.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode T-bet. In a preferred embodiment, the nucleic acid moleculeof the invention comprises the nucleotide sequence shown in SEQ ID NO:1or SEQ ID NO:3. In another embodiment, a nucleic acid molecule of theinvention comprises at least about 700 contiguous nucleotides of SEQ IDNO:1 or at least about 500 contiguous nucleotides of SEQ ID NO:3. In apreferred embodiment, a nucleic acid molecule of the invention comprisesat least about 800, at least about 1000, at east about 1200, at leastabout 1400 or at least about 1600 contiguous nucleotides of SEQ ID NO:1.In another preferred embodiment, a nucleic acid molecule of theinvention comprises at least about 600, at least about 800, at leastabout 1000, at least about 1200, or at least about 1400 contiguousnucleotides of SEQ ID NO:3.

In other embodiments, the nucleic acid molecule has at least 70%identity, more preferably 80% identity, and even more preferably 90%identity with a nucleic acid molecule comprising: at least about 700, atleast about 800, at least about 1000, at east about 1200, at least about1400 or at least about 1600 contiguous nucleotides of SEQ ID NO:1. Inother embodiments, the nucleic acid molecule has at least 70% identity,more preferably 80% identity, and even more preferably 90% nucleotideidentity with a nucleic acid molecule comprising: at least about 600, atleast about 800, at least about 1000, at least about 1200, or at leastabout 1400 contiguous nucleotides of SEQ ID NO:3.

Nucleic acid molecules that differ from SEQ ID NO: 1 or 3 due todegeneracy of the genetic code, and thus encode the same T-bet proteinas that encoded by SEQ ID NO: 1 and 3, are encompassed by the invention.Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having anamino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:4.

In addition, nucleic acid molecules encoding T-bet proteins can beisolated from other sources using standard molecular biology techniquesand the sequence information provided herein. For example, a T-bet DNAcan be isolated from a human genomic DNA library using all or portion ofSEQ ID NO: 1 or 3 as a hybridization probe and standard hybridizationtechniques (e.g., as described in Sambrook, J., et al. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acid moleculeencompassing all or a portion of a T-bet gene can be isolated by thepolymerase chain reaction using oligonucleotide primers designed basedupon the sequence of SEQ ID NO: 1 or 3. For example, mRNA can beisolated from cells (e.g., by the guanidinium-thiocyanate extractionprocedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNAcan be prepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for PCR amplification can bedesigned based upon the nucleotide sequence shown in SEQ ID NO: 1 or 3.A nucleic acid of the invention can be amplified using cDNA or,alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a T-bet nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In addition to the T-bet nucleotide sequence shown in SEQ ID NO: 1 and3, it will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to minor changes in the nucleotide or amino acidsequences of T-bet may exist within a population. Such geneticpolymorphism in the T-bet gene may exist among individuals within apopulation due to natural allelic variation. Such natural allelicvariations can typically result in 1-2% variance in the nucleotidesequence of the a gene. Any and all such nucleotide variations andresulting amino acid polymorphisms in T-bet that are the result ofnatural allelic variation and that do not alter the functional activityof T-bet are intended to be within the scope of the invention.

Nucleic acid molecules corresponding to natural allelic variants of theT-bet DNAs of the invention can be isolated based on their homology tothe T-bet nucleic acid molecules disclosed herein using the human DNA,or a portion thereof, as a hybridization probe according to standardhybridization techniques under high stringency hybridization conditions.Exemplary high stringency conditions include hybridization in ahybridization buffer that contains 6× sodium chloride/sodium citrate(SSC) at a temperature of about 45° C. for several hours to overnight,followed by one or more washes in a washing buffer containing 0.2×SSC,0.1% SDS at a temperature of about 50-65° C. Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the inventionhybridizes under high stringency conditions to a second nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3.Preferably, an isolated nucleic acid molecule of the invention thathybridizes under high stringency conditions to the sequence of SEQ IDNO: of SEQ ID NO:1 or 3. In one embodiment, such a nucleic acid moleculeis at least about 700, 800, 900, 1000, 1200, 1300, 1400, 1500, or 1600nucleotides in length. In another embodiment, such a nucleic acidmolecule and comprises at least about 700, 800, 900, 1000, 1200, 1300,1400, 1500, or 1600 contiguous nucleotides of SEQ ID NO: 1 or at leastabout 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500contiguous nucleotides of SEQ ID NO: 3. Preferably, an isolated nucleicacid molecule corresponds to a naturally-occurring allelic variant of aT-bet nucleic acid molecule.

In addition to naturally-occurring allelic variants of the T-betsequence that may exist in the population, the skilled artisan willfurther appreciate that minor changes may be introduced by mutation intothe nucleotide sequence of SEQ ID NO: 1 or 3, thereby leading to changesin the amino acid sequence of the encoded protein, without altering thefunctional activity of the T-bet protein. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made in the sequence of SEQ ID NO: 1 or 3. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of T-bet (e.g., the sequence of SEQ ID NO: 1 or3) without altering the functional activity of T-bet, such as itsability to interact with DNA or its ability to enhance transcriptionfrom an IFN-γ promoter, whereas an “essential” amino acid residue isrequired for functional activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding T-bet proteins that contain changes in amino acidresidues that are not essential for T-bet activity. Such T-bet proteinsdiffer in amino acid sequence from SEQ ID NO: 2 or 4 yet retain T-betactivity. An isolated nucleic acid molecule encoding a non-naturalvariant of a T-bet protein can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of SEQ ID NO: 1 or 3 such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into SEQ ID NO: 1 or 3 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in T-bet ispreferably replaced with another amino acid residue from the same sidechain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of the T-bet coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened fortheir ability to bind to DNA and/or activate transcription, to identifymutants that retain functional activity. Following mutagenesis, theencoded T-bet mutant protein can be expressed recombinantly in a hostcell and the functional activity of the mutant protein can be determinedusing assays available in the art for assessing T-bet activity (e.g., bymeasuring the ability of the protein to bind to a T-box binding elementpresent in DNA or by measuring the ability of the protein to modulate aTh1 or Th2 phenotype in a T cell.

Another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense to the coding strand of a T-bet mRNA orgene. An antisense nucleic acid of the invention can be complementary toan entire T-bet coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a codingregion of the coding strand of a nucleotide sequence encoding T-bet thatis unique to the T-bet family of proteins or which is unique to a T-betsequence from a particular species. In another embodiment, the antisensenucleic acid molecule is antisense to a noncoding region of the codingstrand of a nucleotide sequence encoding T-bet that is unique to T-betfamily of proteins or which is unique to a T-bet sequence from aparticular species. In preferred embodiments, an antisense molecule ofthe invention comprises at least about 700 contiguous nucleotides of thenoncoding strand of SEQ ID NO: 1, more preferably at least 800, 1000,1200, 1400, or 1600 contiguous nucleotides of the noncoding strand ofSEQ ID NO: 1 or at least about 500 contiguous nucleotides of thenoncoding strand of SEQ ID NO: 3, more preferably at least 600, 800,1000, 1200, or 1400 contiguous nucleotides of the noncoding strand ofSEQ ID NO: 3.

Given the coding strand sequences encoding T-bet disclosed herein (e.g.,SEQ ID NOs: 1 and 3, antisense nucleic acids of the invention can bedesigned according to the rules of Watson and Crick base pairing. Theantisense nucleic acid molecule may be complementary to the entirecoding region of T-bet mRNA, or alternatively can be an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof T-bet mRNA. For example, the antisense oligonucleotide may becomplementary to the region surrounding the translation start site ofT-bet mRNA. An antisense oligonucleotide can be, for example, about 15,20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Alternatively, the antisensenucleic acid can be produced biologically using an expression vectorinto which a nucleic acid has been subcloned in an antisense orientation(i.e., RNA transcribed from the inserted nucleic acid will be of anantisense orientation to a target nucleic acid of interest, describedfurther in the following subsection).

In another embodiment, an antisense nucleic acid of the invention is aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. A ribozymehaving specificity for a T-bet-encoding nucleic acid can be designedbased upon the nucleotide sequence of a T-bet gene disclosed herein. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the base sequence of the active site is complementary to thebase sequence to be cleaved in a T-bet-encoding mRNA. See for exampleCech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, T-bet mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See for example Bartel, D. and Szostak, J. W. (1993) Science261: 1411-1418.

In another embodiment, RNAi can be used to inhibit T-bet expression. RNAinterference (RNAi is a post-transcriptional, targeted gene-silencingtechnique that uses double-stranded RNA (dsRNA) to degrade messenger RNA(mRNA) containing the same sequence as the dsRNA (Sharp, P. A. andZamore, P. D. 287, 2431-2432 (2000); Zamore, P. D., et al. Cell 101,25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197 (1999)). Theprocess occurs when an endogenous ribonuclease cleaves the longer dsRNAinto shorter, 21- or 22-nucleotide-long RNAs, termed small interferingRNAs or siRNAs. The smaller RNA segments then mediate the degradation ofthe target mRNA.

The antisense RNA strand of RNAi can be antisense to at least a portionof the coding region of T-bet or to at least a portion of the 5′ or 3′untranslated region of the T-bet gene. In one embodiment, siRNA duplexesare composed of 21-nt sense and 21-nt antisense strands, paired in amanner to have a 2-nt 3′ overhang. In one embodiment, siRNA sequenceswith TT in the overhang. The target region can be, e.g., 50 to 100 ntdownstream of the start codon, 3′-UTRs may also be targeted. In oneembodiment, a 23-nt sequence motif AA(N19)TT (N, any nucleotide) (SEQ IDNO:15) can be searched for and hits with between about 30-70%G/C-content can be selected. If no suitable sequences are found, thesearch is extended using the motif NA(N21). SiRNAs are preferablychemically synthesized using appropriately protected ribonucleosidephosphoramidites and a conventional DNA/RNA synthesizer. SiRNAs are alsoavailable commercially from, e.g., Dharmacon, Xeragon Inc, Proligo, andAmbion. In one embodiment one or more of the chemistries described abovefor use in antisense RNA can be employed.

Yet another aspect of the invention pertains to isolated nucleic acidmolecules encoding T-bet fusion proteins. Such nucleic acid molecules,comprising at least a first nucleotide sequence encoding a T-betprotein, polypeptide or peptide operatively linked to a secondnucleotide sequence encoding a non-T-bet protein, polypeptide orpeptide, can be prepared by standard recombinant DNA techniques. T-betfusion proteins are described in further detail below in subsection III.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyrecombinant expression vectors, containing a nucleic acid encoding T-bet(or a portion thereof). The expression vectors of the invention comprisea nucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., T-bet proteins, mutant forms ofT-bet proteins, T-bet fusion proteins and the like).

The recombinant expression vectors of the invention can be designed forexpression of T-bet protein in prokaryotic or eukaryotic cells. Forexample, T-bet can be expressed in bacterial cells such as E. coli,insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector may be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors can serve one or more purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification; 4) to provide an epitopetag to aid in detection and/or purification of the protein; and/or 5) toprovide a marker to aid in detection of the protein (e.g., a colormarker using β-galactosidase fusions). Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Recombinantproteins also can be expressed in eukaryotic cells as fusion proteinsfor the same purposes discussed above.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nuc. Acids Res.20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the T-bet expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari. et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

Alternatively, T-bet can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., Sf9 cells) include the pAcseries (Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and the pVLseries (Lucklow, V. A., and Summers, M. D., (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pMex-NeoI, pCDM8 (Seed, B., (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include lymphoid-specific promoters (Calame and Eaton (1988)Adv. Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), the albumin promoter (liver-specific; Pinkert et al. (1987)Genes Dev. 1:268-277), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Moreover, inducible regulatory systems for use in mammalian cells areknown in the art, for example systems in which gene expression isregulated by heavy metal ions (see e.g., Mayo et al. (1982) Cell29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985)Mol. Cell. Biol. 5:1480-1489), heat shock (see e.g., Nouer et al. (1991)in Heat Shock Response, e.d. Nouer, L., CRC, Boca Raton, Fla., pp167-220), hormones (see e.g., Lee et al. (1981) Nature 294:228-232;Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock etal. (1987) Nature 329:734-736; Israel & Kaufman (1989) Nucl. Acids Res.17:2589-2604; and PCT Publication No. WO 93/23431), FK506-relatedmolecules (see e.g., PCT Publication No. WO 94/18317) or tetracyclines(Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; PCTPublication No. WO 94/29442; and PCT Publication No. WO 96/01313).Accordingly, in another embodiment, the invention provides a recombinantexpression vector in which T-bet DNA is operatively linked to aninducible eukaryotic promoter, thereby allowing for inducible expressionof T-bet protein in eukaryotic cells.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to T-bet mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to recombinant host cells intowhich a vector, preferably a recombinant expression vector, of theinvention has been introduced. A host cell may be any prokaryotic oreukaryotic cell. For example, T-bet protein may be expressed inbacterial cells such as E. coli, insect cells, yeast or mammalian cells(such as Chinese hamster ovary cells (CHO) or COS cells). Other suitablehost cells are known to those skilled in the art. Vector DNA can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance tocompounds, such as G418, hygromycin and methotrexate. Nucleic acidencoding a selectable marker may be introduced into a host cell on thesame vector as that encoding T-bet or may be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by compound selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) T-bet protein.Accordingly, the invention further provides methods for producing T-betprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding T-bet has been introduced) in asuitable medium until T-bet is produced. In another embodiment, themethod further comprises isolating T-bet from the medium or the hostcell. In its native form the T-bet protein is an intracellular proteinand, accordingly, recombinant T-bet protein can be expressedintracellularly in a recombinant host cell and then isolated from thehost cell, e.g., by lysing the host cell and recovering the recombinantT-bet protein from the lysate. Alternatively, recombinant T-bet proteincan be prepared as a extracellular protein by operatively linking aheterologous signal sequence to the amino-terminus of the protein suchthat the protein is secreted from the host cells. In this case,recombinant T-bet protein can be recovered from the culture medium inwhich the cells are cultured.

Certain host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichT-bet-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous T-betsequences have been introduced into their genome or homologousrecombinant animals in which endogenous T-bet sequences have beenaltered. Such animals are useful for studying the function and/oractivity of T-bet and for identifying and/or evaluating modulators ofT-bet activity. Accordingly, another aspect of the invention pertains tononhuman transgenic animals which contain cells carrying a transgeneencoding a T-bet protein or a portion of a T-bet protein. In asubembodiment, of the transgenic animals of the invention, the transgenealters an endogenous gene encoding an endogenous T-bet protein (e.g.,homologous recombinant animals in which the endogenous T-bet gene hasbeen functionally disrupted or “knocked out”, or the nucleotide sequenceof the endogenous T-bet gene has been mutated or the transcriptionalregulatory region of the endogenous T-bet gene has been altered).

A transgenic animal of the invention can be created by introducingT-bet-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. The T-bet nucleotide sequence ofSEQ ID NO: 1 or 3 can be introduced as a transgene into the genome of anon-human animal. Intronic sequences and polyadenylation signals canalso be included in the transgene to increase the efficiency ofexpression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to the T-bet transgene to direct expression ofT-bet protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the T-bet transgene in its genomeand/or expression of T-bet mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding T-bet can further be bred to other transgenic animalscarrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a T-bet gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the endogenous T-bet gene. In one embodiment, ahomologous recombination vector is designed such that, upon homologousrecombination, the endogenous T-bet gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous T-bet gene replacedby the T-bet gene. In the homologous recombination vector, the alteredportion of the T-bet gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the T-bet gene to allow for homologous recombination tooccur between the exogenous T-bet gene carried by the vector and anendogenous T-bet gene in an embryonic stem cell. The additional flankingT-bet nucleic acid is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector(see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced T-bet gene has homologously recombinedwith the endogenous T-bet gene are selected (see e.g., Li, E. et al.(1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

In addition to the foregoing, the skilled artisan will appreciate thatother approaches known in the art for homologous recombination can beapplied to the instant invention. Enzyme-assisted site-specificintegration systems are known in the art and can be applied to integratea DNA molecule at a predetermined location in a second target DNAmolecule. Examples of such enzyme-assisted integration systems includethe Cre recombinase-lox target system (e.g., as described in Baubonis,W. and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige, S.and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89:7905-7909) and theFLP recombinase-FRT target system (e.g., as described in Dang, D. T. andPerrimon, N. (1992) Dev. Genet. 13:367-375; and Fiering, S. et al.(1993) Proc. Natl. Acad. Sci. USA 90:8469-8473). Tetracycline-regulatedinducible homologous recombination systems, such as described in PCTPublication No. WO 94/29442 and PCT Publication No. WO 96/01313, alsocan be used.

In another embodiment, transgenic animals can be made in which T-bet isexpressed in all T cells, e.g., using the CD4 enhancer (Zheng, W-P. &Flavell, R. A. 1997. Cell 89, 587). Recent work suggests the CD2enhancer can also be used. In fact, it is more powerful in achievinghigh level expression in T cells, expression is not variegated andtransgene expression is copy number-dependent (Zhumabekov, T., et al.1995. J. Immunol. Meth. 185, 133; Sharp, L. L., et al. 1997. Immunity 7,609). Mice with high level expression of T-bet RNA (using the humangrowth hormone intron as a probe to distinguish transgene driven T-betRNA from endogenous T-bet) can be identified by screening adequatenumbers of founders.

In another approach, a dominant repressor transgenic can be created. Forexample, a dominant-repressor T-bet can be made by using the proximallck enhancer (Alberola-Ila, J., et al. 1996 J. Exp. Med. 184, 9) drivinga fusion of T-bet and engrailed can be made (Taylor, D., 1996. GenesDev. 10, 2732; Li, J., Thurm, H., et al. 1997. Proc. Natl. Acad. Sci.USA 94, 10885). This construct specifically represses T-bettransactivation of a multimerized T-bet reporter and does not affectNFAT-dependent reporter transactivation.

Alternatively, null mutations can be generated by targeted mutagenesisin ES cells (Ranger, A. M., et al. 1998. Nature 392, 186; Hodge, M. R.,et al. 1996. Immunity 4:1., 144; Grusby, M. J., et al. 1991. Science253, 1417; Reimold, A. M., et al. 1996. Nature 379: 262; Kaplan, M. H.,1996. Immunity: 313; Kaplan, M. H., et al. 1996. Nature 382, 174;Smiley, S. T., et al. 1997. Science 275, 977). For example usingtechniques which are known in the art, a genomic T-bet clone can beisolated from a genomic library, the intron-exon organizationdelineated, and a targeting construct in the cre-lox vector (seediscussion below) created which should delete the first exon and 450 bpof upstream promoter sequence. This construct can be electroporated intoan ES cell line, and double compound resistant (e.g., neomycin,gancyclovir) clones identified by Southern blot analysis. Clones bearinghomologous recombinant events in the T-bet locus can then be identifiedand injected into blastocysts obtained from day 3.5 BALB/c pregnantmice. Chimeric mice can then be produced and mated to wildtype BALB/cmice to generate germline transmission of the disrupted T-bet gene.

In another embodiment, implantation into RAG2-deficient blastocysts(Chen, J., et al. 1993. Proc. Natl. Acad. Sci. USA 90, 4528) or thecre-lox inducible deletion approach can be used to develop mice that arelacking T-bet only in the immune system. For example, the targetingconstruct can be made in the cre-lox vector. The blastocystcomplementation system has been used to study NFATc, an embryonic lethalphenotype (Ranger, A. M., et al. 1998. Immunity 8:125). This approachrequires disrupting the T-bet gene on both chromosomes in ES cells,which can be accomplished, e.g., by using a mutant neomycin gene andraising the concentration of G418 in the ES cultures, as described(Chen, J., 1993. Proc. Natl. Acad. Sci. USA 90; 4528) or by flanking theneo gene with cre-lox sites. To disrupt the second allele, the neomycingene can be deleted by transfecting the ES clone with the crerecombinase, and then the ES clone can be retransfected with the sametargeting construct to select clones with T-bet deletions on bothalleles. A third transfection with cre-recombinase yields the desireddoubly-deficient ES cells. Such doubly targeted ES cells are thenimplanted into RAG2 blastocysts and the lymphoid organs of the chimericmice thus generated will be entirely colonized by the transferred EScells. This allows assessment of the effect of the absence of T-bet oncells of the lymphoid system without affecting other organ systems wherethe absence of T-bet might cause lethality.

The conditional ablation approach employing the cre-lox system can alsobe used. Briefly, a targeting construct is generated in which loxrecombination sequences are placed in intronic regions flanking theexons to be deleted. This construct is then transfected into ES cellsand mutant mice are generated as above. The resulting mutant mice arethen mated to mice transgenic for the cre recombinase driven by aninducible promoter. When cre is expressed, it induces recombinationbetween the introduced lox sites in the T-bet gene, thus effectivelydisrupting gene function. The key feature of this approach is that genedisruption can be induced in the adult animal at will by activating thecre recombinase.

A tissue-specific promoter can be used to avoid abnormalities in organsoutside the immune system. The cre-expressing transgene may be driven byan inducible promoter. Several inducible systems are now being used incre-lox recombination strategies, the most common being the tetracyclineand ecdysone systems. A tissue-specific inducible promoter can be usedif there is embryonic lethality in the T-bet null mouse.

An alternative approach is to generate a transgenic mouse harboring aregulated T-bet gene (for example using the tetracycline off promoter;e.g., St-Onge, et al. 1996. Nuc. Acid Res. 24, 3875-3877) and then breedthis transgenic to the T-bet deficient mouse. This approach permitscreation of mice with normal T-bet function; tetracycline can beadministered to adult animals to induce disruption of T-bet function inperipheral T cells, and then the effect of T-bet deficiency can beexamined over time. Repeated cycles of provision and then removal ofcompound (tetracycline) permits turning the T-bet gene on and off atwill.

III. Isolated T-Bet Proteins and Anti-T-Bet Antibodies

Another aspect of the invention pertains to isolated T-bet proteins.Preferably, the T-bet protein comprises the amino acid sequence encodedby SEQ ID NO:1 or 3. In another preferred embodiment, the proteincomprises the amino acid sequence of SEQ ID NO: 2 or 4. In otherembodiments, the protein has at least 60% amino acid identity, morepreferably 70% amino acid identity, more preferably 80%, and even morepreferably, 90% or 95% amino acid identity with the amino acid sequenceshown in SEQ ID NO: 2 or 4.

In other embodiments, the invention provides isolated portions of theT-bet protein. For example, the invention further encompasses anamino-terminal portion of T-bet that includes a T-box domain. In variousembodiments, this amino terminal portion encompasses at least aminoacids 138-327 of human T-bet or at least amino acids 137-326 of mouseT-bet. Another isolated portion of T-bet provided by the invention is aportion encompassing a tyrosine phosphorylation site. This portioncomprises at least about 20, at least about 50, at least about 100, orat least about 200 amino acids of T-bet and includes at least aminoacids Tyr 76, Tyr 119, and/or Tyr 531 of human T-bet or amino acids Tyr525 of murine T-bet. Yet another isolated portion of T-bet providedherein is a portion encompassing a nuclear localization sequence shownin amino acids 498-501 of human T-bet or 493-496 of murine T-bet.

T-bet proteins of the invention are preferably produced by recombinantDNA techniques. For example, a nucleic acid molecule encoding theprotein is cloned into an expression vector (as described above), theexpression vector is introduced into a host cell (as described above)and the T-bet protein is expressed in the host cell. The T-bet proteincan then be isolated from the cells by an appropriate purificationscheme using standard protein purification techniques. Alternative torecombinant expression, a T-bet polypeptide can be synthesizedchemically using standard peptide synthesis techniques. Moreover, nativeT-bet protein can be isolated from cells (e.g., from T cells), forexample by immunoprecipitation using an anti-T-bet antibody.

The present invention also pertains to variants of the T-bet proteinswhich function as either T-bet agonists (mimetics) or as T-betantagonists. Variants of the T-bet proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a T-betprotein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. In one embodiment, treatment of asubject with a variant having a subset of the biological activities ofthe naturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of theT-bet protein. In one embodiment, the invention pertains to derivativesof T-bet which may be formed by modifying at least one amino acidresidue of T-bet by oxidation, reduction, or other derivatizationprocesses known in the art.

In one embodiment, variants of a T-bet protein which function as eitherT-bet agonists (mimetics) or as T-bet antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a T-bet protein for T-bet protein agonist or antagonist activity. Inone embodiment, a variegated library of T-bet variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of T-bet variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential T-bet sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of T-bet sequences therein. There are avariety of methods which can be used to produce libraries of potentialT-bet variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential T-bet sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A., 1983,Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323;Itakura et al., 1984, Science 198:1056; Ike et al., 1983, Nucleic AcidRes. 11:477).

In addition, libraries of fragments of a T-bet protein coding sequencecan be used to generate a variegated population of T-bet fragments forscreening and subsequent selection of variants of a T-bet protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of a T-bet coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the T-bet protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of T-bet proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify T-bet variants (Arkin and Yourvan, 1992, Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering6(3):327-331).

The invention also provides T-bet fusion proteins. As used herein, aT-bet “fusion protein” comprises a T-bet polypeptide operatively linkedto a polypeptide other than T-bet. A “T-bet polypeptide” refers to apolypeptide having an amino acid sequence corresponding to T-betprotein, or a peptide fragment thereof which is unique to T-bet proteinwhereas a “polypeptide other than T-bet” refers to a polypeptide havingan amino acid sequence corresponding to another protein. Within thefusion protein, the term “operatively linked” is intended to indicatethat the T-bet polypeptide and the other polypeptide are fused in-frameto each other. The other polypeptide may be fused to the N-terminus orC-terminus of the T-bet polypeptide. For example, in one embodiment, thefusion protein is a GST-T-bet fusion protein in which the T-betsequences are fused to the C-terminus of the GST sequences. In anotherembodiment, the fusion protein is a T-bet-HA fusion protein in which theT-bet nucleotide sequence is inserted in a vector such as pCEP4-HAvector (Herrscher, R. F. et al. (1995) Genes Dev. 9:3067-3082) such thatthe T-bet sequences are fused in frame to an influenza hemagglutininepitope tag. Such fusion proteins can facilitate the purification ofrecombinant T-bet.

Preferably, a T-bet fusion protein of the invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide or an HA epitope tag). A T-bet-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the T-bet protein.

An isolated T-bet protein, or fragment thereof, can be used as animmunogen to generate antibodies that bind specifically to T-bet usingstandard techniques for polyclonal and monoclonal antibody preparation.The T-bet protein can be used to generate antibodies. For example,polyclonal antisera, can be produced in rabbits using full-lengthrecombinant bacterially produced T-bet as the immunogen. This sameimmunogen can be used to produce mAb by immunizing mice and removingspleen cells from the immunized mice. Spleen cells from mice mounting animmune response to T-bet can be fused to myeloma cells, e.g., SP2/O-Ag14myeloma. As described in the appended examples, this methods were usedto make polyclonal and monoclonal antibodies which bind to T-bet. In oneembodiment, the antibodies can be produced in an animal that does notexpress T-bet, such as a T-bet knock-out animal. In another embodiment,the antibodies can be generated in a non-human animal having a specificgenetic background, e.g., as achieved by backcrossing.

Alternatively, an antigenic peptide fragment of T-bet can be used as theimmunogen. An antigenic peptide fragment of T-bet typically comprises atleast 8 amino acid residues of the amino acid sequence shown in SEQ IDNO: 2 or 4 and encompasses an epitope of T-bet such that an antibodyraised against the peptide forms a specific immune complex with T-bet.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues. Preferred epitopes encompassed by the antigenicpeptide are regions of T-bet that are located on the surface of theprotein, e.g., hydrophilic regions, and that are unique to T-bet. In oneembodiment such epitopes can be specific for T-bet proteins from onespecies, such as mouse or human (i.e., an antigenic peptide that spans aregion of T-bet that is not conserved across species is used asimmunogen; such non conserved residues can be determined using analignment such as that provided herein). A standard hydrophobicityanalysis of the T-bet protein can be performed to identify hydrophilicregions.

A T-bet immunogen typically is used to prepare antibodies by immunizinga suitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexamples, recombinantly expressed T-bet protein or a chemicallysynthesized T-bet peptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic T-bet preparation induces a polyclonal anti-T-bet antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-T-betantibodies. Polyclonal anti-T-bet antibodies can be prepared asdescribed above by immunizing a suitable subject with a T-bet immunogen.The anti-T-bet antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized T-bet. If desired, theantibody molecules directed against T-bet can be isolated from themammal (e.g., from the blood) and further purified by well knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the anti-T-betantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al.(1981) J. Immunol 127:539-46; Brown et al. (1980) J Biol Chem255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982)Int. J. Cancer 29:269-75), the more recent human B cell hybridomatechnique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridomatechnique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977)Somatic Cell Genet., 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a T-bet immunogen as described above, andthe culture supernatants of the resulting hybridoma cells are screenedto identify a hybridoma producing a monoclonal antibody that bindsspecifically to T-bet.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-T-bet monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinary skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines may be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from the American TypeCulture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind T-bet, e.g., using a standard ELISA assay.

Using such methods several antibodies to T-bet have been generated. Bothmonoclonal and polyclonal antibodies were generated against full-lengthrecombinant bacterially produced T-bet protein. The 3D10 antibody is ofthe IgG subtype and the 4B10 antibody was produced by fusion of mousespleen cells to the SP2/0-Ag14 myeloma and is of the IgG subtype. The39D antibody recognizes both human and murine T-bet.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-T-bet antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with T-bet to thereby isolateimmunoglobulin library members that bind T-bet. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clarkson etal. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580;Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133-4137; Barbas et al. (1991) PNAS88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-T-bet antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Patent Publication PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al. U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal. (1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443;Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

In another embodiment, fully human antibodies can be made usingtechniques that are known in the art. For example, fully humanantibodies against a specific antigen can be prepared by administeringthe antigen to a transgenic animal which has been modified to producesuch antibodies in response to antigenic challenge, but whose endogenousloci have been disabled. Exemplary techniques that can be used to makeantibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592;6,420,140. Other techniques are known in the art.

An anti-T-bet antibody (e.g., monoclonal antibody) can be used toisolate T-bet by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-T-bet antibody can facilitate thepurification of natural T-bet from cells and of recombinantly producedT-bet expressed in host cells. Moreover, an anti-T-bet antibody can beused to detect T-bet protein (e.g., in a cellular lysate or cellsupernatant). Detection may be facilitated by coupling (i.e., physicallylinking) the antibody to a detectable substance. Accordingly, in oneembodiment, an anti-T-bet antibody of the invention is labeled with adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S or ³H.

Yet another aspect of the invention pertains to anti-T-bet antibodiesthat are obtainable by a process comprising:

-   -   (a) immunizing an animal with an immunogenic T-bet protein, or        an immunogenic portion thereof unique to T-bet protein; and    -   (b) isolating from the animal antibodies that specifically bind        to a T-bet protein.

Methods for immunization and recovery of the specific anti-T-betantibodies are described further above.

In yet another aspect, the invention pertains to T-bet intrabodies.Intrabodies are intracellularly expressed antibody constructs, usuallysingle-chain Fv (scFv) antibodies directed against a target inside acell, e.g. an intracellular protein such as T-bet (Graus-Porta, D. etal. (1995) Mol. Cell Biol. 15(1):182-91). For example, an intrabody(e.g., and scFv) can contain the variable region of the heavy and thelight chain, linked by a flexible linker and expressed from a singlegene. The variable domains of the heavy and the light chain contain thecomplementarity determining regions (CDRs) of the parent antibody, i.e.,the main antigen binding domains, which determine the specificity of thescFvs. The scFv gene can be transferred into cells, where scFv proteinexpression can modulate the properties of its target, e.g., T-bet.Accordingly, in one embodiment, the invention provides a method forusing such T-bet intrabodies to prevent T-bet activity in cells, forexample, in an in vivo or ex vivo approach, for which the cells aremodified to express such intrabodies. In a particular embodiment, theT-bet intrabodies of the invention can be used to directly inhibit T-betactivity. In another embodiment, the T-bet intrabodies can be used toinhibit the interaction of T-bet and a protein with which T-betinteracts. Thus, the T-bet intrabodies of the invention are useful inmodulating signaling pathways in which T-bet is involved.

The T-bet intrabodies can be prepared using techniques known in the art.For example, phage display technology can be used to isolate scFvs fromlibraries (Lowman, H B et al. (1991) Biochemistry 30(10): 832-8). Toselect scFvs binding to a particular antigen, the scFvs are fused to acoat protein, typically pIII (g3p) of filamentous M13 phage. An scFv onthe phage that binds an immobilized antigen is enriched duringconsecutive cycles of binding, elution and amplification. In anotherexample, ribosome display can used to prepare T-bet intrabodies (Hanes,J. et al. (1997) Proc. Natl. Acad. Sci. 94(1): 937-44). Ribosome displayis an in vitro method that links the peptide directly to the geneticinformation (mRNA). An scFv CDNA library is expressed in vitro using atranscription translation system. The translated ScFvs are stalled tothe ribosome linked to the encoding mRNA. The scFv is then bound to theimmobilized antigen and unspecific ribosome complexes are removed byextensive washes. The remaining complexes are eluted and the RNA isisolated, reverse transcribed to cDNA and subsequently re-amplified byPCR. In yet another example, a Protein Fragment Complementation Assay(PCA) can be used to prepare T-bet intrabodies of the invention(Pelletier, J N et al. (1998) Proc. Natl. Acad. Sci. 95(12): 141-6.)This is a cellular selection procedure based on the complementation of amutant dihydrofolate reductase (DHFR) in E. coli by the mouse protein(mDHFR). The murine DHFR is dissected into two pairs, which areexpressed as fusion proteins with potentially interacting peptides. Theinteraction of the fusion proteins restores the enzymatic activity ofmDHFR, and thus bacterial proliferation. Only a specific interaction ofantibody and antigen allows the functional complementation of DHFR whichmakes the system amenable for the selection of scFvs (Mossner, E. et al.(2001) J Mol. Biol. 308: 115-22).

IV. Pharmaceutical Compositions

T-bet modulators of the invention (e.g., T-bet inhibitory or stimulatoryagents, including T-bet nucleic acid molecules, proteins, antibodies, orcompounds described herein or identified as modulators of T-betactivity) can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the modulatoryagent and a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. For example,solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

In one embodiment, compositions comprising T-bet modulating agents cancomprise a second agent which is useful in modulating a cellularresponse affected by T-bet. For example, in one embodiment, an agentwhich downmodulates T-bet activity may be administered in combinationwith a second agent that downmodulates a cellular immune response. Suchagents may be administered as part of the same pharmaceuticalcomposition as the T-bet modulating agent or may be formulated forseparate administration.

V. Methods of the Invention

Diagnostic Assays/Prognostic Assays

Another aspect of the invention pertains to methods of using the variousT-bet compositions of the invention. For example, the invention providesa method for detecting the presence of T-bet activity in a biologicalsample. The method involves contacting the biological sample with anagent capable of detecting T-bet activity, such as T-bet protein orT-bet mRNA, such that the presence of T-bet activity is detected in thebiological sample.

A preferred agent for detecting T-bet mRNA is a labeled nucleic acidprobe capable of specifically hybridizing to T-bet mRNA. The nucleicacid probe can be, for example, the T-bet DNA of SEQ ID NO: 1 or 3, suchas an oligonucleotide of at least about 500, 600, 800, 900, 1000, 1200,1400, or 1600 nucleotides in length and which specifically hybridizesunder stringent conditions to T-bet mRNA.

A preferred agent for detecting T-bet protein is a labeled antibodycapable of binding to T-bet protein. Antibodies can be polyclonal, ormore preferably, monoclonal. An intact antibody, or a fragment thereof(e.g., Fab or F(ab′)₂) can be used. The term “labeled”, with regard tothe probe or antibody, is intended to encompass direct labeling of theprobe or antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids. For example,techniques for detection of T-bet mRNA include Northern hybridizationsand in situ hybridizations. Techniques for detection of T-bet proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence.

Such assays are useful in detecting syndromes characterized bydevelopmental defects. For example, mutations in the human T-box genesTBX5 and TBX3 (orthologs of mouse Tbx5 and Tbx3) are responsible for theautosomal dominant genetic diseases Holt-Oram syndrome and ulnar-mammarysyndrome respectively (Bamshad, M., et al. 1997. Nature Genetics 16:311; Basson, C. T., et al. 1997. Nature Genetics 15:30; Li, Q. Y., etal. 1997. Nature Genetics 15: 21; Spranger, S., et al. 1997. J. Med.Genet. 3:978). These syndromes are characterized by developmentaldefects and might have been predicted by the patterns of expression ofTbx5 and Tbx3 respectively. Holt-Oram syndrome affects the heart andupper limbs while ulnar-mammary syndrome affects limb, apocrine gland,tooth and genital development. Both syndromes are characterized bydevelopmental defects and might have been predicted by the patterns ofexpression of Tbx5 and Tbx3 respectively. The mutations in thesepatients involve only one allele of the T-box gene—thus it has beenpostulated that haploinsufficiency of Tbx3 and Tbx 5 cause these twodiseases. Recently it has been demonstrated that provision of Tbx4 andTbx5 to developing chick embryos controls limb bud identity(Rodriguez-Esteban et al., 1999; Takeuchi et al., 1999). Thesediscoveries emphasize the critical importance of this family invertebrate development.

In addition, the existence of T gene homologs in many species providesstrong evidence for its function as a transcription factor thatregulates a set of as yet unknown target genes involved in mesodermdevelopment. The recent prominence of the T-box family arises from itsclear importance in diverse developmental processes, exemplified mostdramatically by the T-box mutations in human disease. The generation ofmature T cells from thymocyte stem cells and of differentiated Th cellsfrom naive precursors can also be viewed as tightly regulateddevelopmental processes. This discovery that T-bet is responsible forthe development of the Th1 lineage demonstrates an important role forthis newest T-box family member in the lymphoid system.

Screening Methods

The invention further provides methods for identifying compounds thatmodulate the expression of T-bet (the amount of T-bet in a cell) and/oractivity of a T-bet polypeptide (the ability of T-bet to propagatesignals in a cell that are brought about as the result of extracellularinfluences on the cell. For example, the invention provides a method foridentifying a compound that modulates the activity of a T-betpolypeptide, comprising

-   -   providing an indicator composition that comprises a T-bet        polypeptide;    -   contacting the indicator composition with a test compound; and    -   determining the effect of the test compound on the activity of        the T-bet polypeptide in the indicator composition to thereby        identify a compound that modulates the activity of a T-bet        polypeptide.

The activity of a T-bet molecule can be detected in a variety of ways.T-bet is a transcription factor and, therefore, has the ability to bindto DNA and to regulate expression of genes, e.g., cytokine genes astaught in the Examples. Accordingly, specific embodiments of thescreening methods of the invention exploit the ability of T-betpolypeptides to bind to DNA; (e.g., IL-2 or IFN-γ promoter); to regulategene expression (e.g., regulate expression of a Th1-associated cytokinegene, e.g., by repressing the IL-2 gene, transactivating the IFN-γ geneor to regulate the expression of a Th2-associated cytokine gene, e.g.,by repressing the IL-4 gene or the IL-10 gene, or to regulate theexpression of other genes, (e.g., TGF-β or Toll-like receptor genes,such as TLR6)). In other embodiments, the ability of T-bet to modulateother cellular responses can be measured, for example, as is shownherein, T-bet regulates, inter alia: immunoglobulin class switching; thegeneration of CD8 effector/memory cells, peripheral tolerance (e.g., byregulating the number of Tr cells present); drives naïve CD4+ cellstowards a Th1 cytokine secretion profile, and/or redirects polarized Th2cells into the Th1 pathway. Accordingly, any of these or other sucheffects of T-bet on cells can be used as readouts in screening forcompounds that modulate the expression and/or activity of T-bet.

In a preferred embodiment of the screening assays of the invention, theindicator composition comprises an indicator cell, wherein saidindicator cell comprises: (i) the a T-bet polypeptide and (ii) areporter gene responsive to the T-bet polypeptide. Preferably, theindicator cell contains:

-   -   i) a recombinant expression vector encoding the T-bet; and    -   ii) a vector comprising regulatory sequences of a Th1-associated        cytokine gene operatively linked a reporter gene; and said        method comprising:

a) contacting the indicator cell with a test compound;

b) determining the level of expression of the reporter gene in theindicator cell in the presence of the test compound; and

c) comparing the level of expression of the reporter gene in theindicator cell in the presence of the test compound with the level ofexpression of the reporter gene in the indicator cell in the absence ofthe test compound to thereby identify a compound that modulates theexpression and/or activity of T-bet.

In another preferred embodiment, the indicator composition comprises apreparation of: (i) a T-bet polypeptide and (ii) a DNA molecule to whichthe T-bet binds, and

said method comprising:

a) contacting the indicator composition with a test compound;

b) determining the degree of interaction of the T-bet polypeptide andthe DNA molecule in the presence of the test compound; and

c) comparing the degree of interaction of the T-bet and the DNA moleculein the presence of the test compound with the degree of interaction ofthe T-bet polypeptide and the DNA molecule in the absence of the testcompound to thereby identify a compound that modulates the expressionand/or activity of T-bet.

Preferably, the DNA molecule to which T-bet binds comprises a T-boxbinding sequence. Such sequences are known in the art, see, e.g., (Szaboet al. 2000. Cell 100:655-669).

In another preferred embodiment, the method identifies polypeptides thatinteract with T-bet. In this embodiment,

the indicator composition is an indicator cell, which indicator cellcomprises:

-   -   i) a reporter gene operably linked to a transcriptional        regulatory sequence; and    -   ii) a first chimeric gene which encodes a first fusion protein,        said first fusion protein including T-bet;

the test compound comprises a library of second chimeric genes, whichlibrary encodes second fusion proteins;

expression of the reporter gene being sensitive to interactions betweenthe first fusion protein, the second fusion protein and thetranscriptional regulatory sequence; and

wherein the effect of the test compound on T-bet in the indicatorcomposition is determined by determining the level of expression of thereporter gene in the indicator cell to thereby identify a test compoundcomprising a polypeptide that interacts with T-bet.

In a preferred embodiment, the library of second chimeric genes isprepared from cDNA library from Th2 cells.

In a preferred embodiment of the screening assays of the invention, oncea test compound is identified as modulating the expression and/oractivity of T-bet, the effect of the test compound on a cellularresponse modulated by T-bet is then tested. Accordingly, the screeningmethods of the invention can further comprise determining the effect ofthe compound on an immune response to thereby identify a compound thatmodulates such a cellular response, e.g., a T cell or B cell response.In one embodiment, the effect of the compound on an immune response isdetermined by determining the effect of the compound on expression of aTh1-associated cytokine gene, such as an interferon-γ gene. As usedherein, the term “Th1-associated cytokine” is intended to refer to acytokine that is produced preferentially or exclusively by Th1 cellsrather than by Th2 cells. Examples of Th1-associated cytokines includeIFN-γ, IL-2, TNF, and lymphotoxin (LT). In another embodiment, theeffect of the compound of interest on an immune response is determinedby determining the effect of the compound on development of T helpertype 1 (Th1) or T helper type 2 (Th2) cells.

In one embodiment, the invention provides methods for identifyingmodulators, i.e., candidate or test compounds or agents (e.g., enzymes,peptides, peptidomimetics, small molecules, ribozymes, or T-betantisense molecules) which bind to T-bet polypeptides; have astimulatory or inhibitory effect on T-bet expression; T-bet processing;T-bet post-translational modification (e.g., glycosylation,ubiquitinization, or phosphorylation); or T-bet activity; or have astimulatory or inhibitory effect on the expression, processing oractivity of a T-bet target molecule.

The indicator composition can be a cell that expresses the T-betpolypeptide, for example, a cell that naturally expresses or, morepreferably, a cell that has been engineered to express the polypeptideby introducing into the cell an expression vector encoding thepolypeptide. Alternatively, the indicator composition can be a cell-freecomposition that includes the polypeptide (e.g., a cell extract from aT-bet expressing cell or a composition that includes purified T-betpolypeptide, either natural or recombinant).

Compounds identified using the assays described herein may be useful fortreating disorders associated with aberrant T-bet expression,processing, post-translational modification, or activity, e.g.,modulation of T cell lineage commitment, modulating the production ofcytokines, modulating TGF-β mediated signaling, modulating theJak1/STAT-1 pathway, modulating IgG class switching and modulating Blymphocyte function.

Conditions that may benefit from modulation of T-bet include autoimmunedisorders including: diabetes mellitus, rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis, multiplesclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmunethyroiditis, atopic dermatitis and eczematous dermatitis, psoriasis,Sjögren's Syndrome, alopecia areata, allergic responses due to arthropodbite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, allergic asthma, cutaneouslupus erythematosus, scleroderma, vaginitis, proctitis, compounderuptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, experimental allergicencephalomyelitis (EAE), and interstitial lung fibrosis

The subject screening assays can be performed in the presence or absenceof other agents. For example, the subject assays can be performed in thepresence various agents that modulate the activation state of the cellbeing screened. For example, in one embodiment, agents that transducesignals via the T cell receptor are included. In another embodiment, acytokine or an antibody to a cytokine receptor is included.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell-free assay, and the abilityof the agent to modulate the expression and/or activity of a T-betpolypeptide can be confirmed in vivo, e.g., in an animal such as ananimal model for multiple sclerosis (EAE), rheumatoid arthritis, orinfection.

Moreover, a T-bet modulator identified as described herein (e.g., adominant negative T-bet molecule, a T-bet nucleic acid or polypeptidemolecule, an antisense T-bet nucleic acid molecule, a T-bet-specificantibody, or a small molecule) can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with sucha modulator. Alternatively, a T-bet modulator identified as describedherein can be used in an animal model to determine the mechanism ofaction of such a modulator.

In another embodiment, it will be understood that similar screeningassays can be used to identify compounds that indirectly modulate aT-bet expression and/or activity, e.g., by performing screening assayssuch as those described above, but employing molecules with which T-betinteracts, i.e., molecules that act either upstream or downstream ofT-bet in a signal transduction pathway, such as a Tec kinase.

Accordingly, as described below, the invention provides a screeningassay for identifying compounds that modulate the interaction of T-betand a T-box binding region (e.g., a cytokine gene regulatory region,such as an IL-2 or IFN-γ gene regulatory region). Assays are known inthe art that detect the interaction of a DNA binding protein with atarget DNA sequence (e.g., electrophoretic mobility shift assays, DNAseI footprinting assays and the like). By performing such assays in thepresence and absence of test compounds, these assays can be used toidentify compounds that modulate (e.g., inhibit or enhance) theinteraction of the DNA binding protein with its target DNA sequence.

The cell based and cell free assays of the invention are described inmore detail below.

A. Cell Based Assays

The indicator compositions of the invention can be a cell that expressesa T-bet polypeptide (or non-T-bet polypeptide such as IFN-γ), forexample, a cell that naturally expresses endogenous T-bet or, morepreferably, a cell that has been engineered to express an exogenousT-bet polypeptide by introducing into the cell an expression vectorencoding the polypeptide. Alternatively, the indicator composition canbe a cell-free composition that includes T-bet or a non-T-betpolypeptide such as IFN-γ (e.g., a cell extract from a T-bet-expressingcell or a composition that includes purified T-bet, either natural orrecombinant polypeptide).

Compounds that modulate expression and/or activity of T-bet (or anon-T-bet polypeptide that acts upstream or downstream of T-bet) can beidentified using various “read-outs.”

For example, an indicator cell can be transfected with a T-betexpression vector, incubated in the presence and in the absence of atest compound, and the effect of the compound on the expression of themolecule or on a biological response regulated by T-bet can bedetermined. The biological activities of T-bet include activitiesdetermined in vivo, or in vitro, according to standard techniques. AT-bet activity can be a direct activity, such as an association with aT-bet-target molecule (e.g., a nucleic acid molecule to which T-betbinds such as the transcriptional regulatory region of a cytokine gene)or a polypeptide such as a kinase. Alternatively, a T-bet activity is anindirect activity, such as a cellular signaling activity occurringdownstream of the interaction of the T-bet polypeptide with a T-bettarget molecule or a biological effect occurring as a result of thesignaling cascade triggered by that interaction. For example, biologicalactivities of T-bet described herein include: modulation of T celllineage commitment, modulating the production of cytokines, modulatingTGF-β mediated signaling, modulating the Jak1/STAT-1 pathway, modulatingIgG class switching and modulating B lymphocyte function. The variousbiological activities of T-bet can be measured using techniques that areknown in the art. Exemplary techniques are described in more detail inthe Examples.

To determine whether a test compound modulates T-bet expression, invitro transcriptional assays can be performed. To perform such an assay,the full length promoter and enhancer of T-bet can be operably linked toa reporter gene such as chloramphenicol acetyltransferase (CAT) orluciferase and introduced into host cells.

As used interchangeably herein, the terms “operably linked” and“operatively linked” are intended to mean that the nucleotide sequenceis linked to a regulatory sequence in a manner which allows expressionof the nucleotide sequence in a host cell (or by a cell extract).Regulatory sequences are art-recognized and can be selected to directexpression of the desired polypeptide in an appropriate host cell. Theterm regulatory sequence is intended to include promoters, enhancers,polyadenylation signals and other expression control elements. Suchregulatory sequences are known to those skilled in the art and aredescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell to be transfected and/or the type and/oramount of polypeptide desired to be expressed.

A variety of reporter genes are known in the art and are suitable foruse in the screening assays of the invention. Examples of suitablereporter genes include those which encode chloramphenicolacetyltransferase, beta-galactosidase, alkaline phosphatase orluciferase. Standard methods for measuring the activity of these geneproducts are known in the art.

A variety of cell types are suitable for use as indicator cells in thescreening assay. Preferably a cell line is used which does not normallyexpress T-bet, such as a Th2 cell clone or a cell from a knock outanimal. Nonlymphoid cell lines can also be used as indicator cells, suchas the HepG2 hepatoma cell line. Yeast cells also can be used asindicator cells.

Cells for use in the subject assays include both eukaryotic andprokaryotic cells. For example, in one embodiment, a cell is a bacterialcell. In another embodiment, a cell is a fungal cell, such as a yeastcell. In another embodiment, a cell is a vertebrate cell, e.g., an aviancell or a mammalian cell (e.g., a murine cell, or a human cell).

In one embodiment, the level of expression of the reporter gene in theindicator cell in the presence of the test compound is higher than thelevel of expression of the reporter gene in the indicator cell in theabsence of the test compound and the test compound is identified as acompound that stimulates the expression of T-bet. In another embodiment,the level of expression of the reporter gene in the indicator cell inthe presence of the test compound is lower than the level of expressionof the reporter gene in the indicator cell in the absence of the testcompound and the test compound is identified as a compound that inhibitsthe expression of T-bet.

In one embodiment, the invention provides methods for identifyingcompounds that modulate cellular responses in which T-bet is involved.

The ability of a test compound to modulate T-bet binding to a targetmolecule or to bind to T-bet can also be determined. Determining theability of the test compound to modulate T-bet binding to a targetmolecule (e.g., a binding partner) can be accomplished, for example, bycoupling the T-bet target molecule with a radioisotope, enzymatic orfluorescent label such that binding of the T-bet target molecule toT-bet can be determined by detecting the labeled T-bet target moleculein a complex. Alternatively, T-bet could be coupled with a radioisotope,enzymatic or fluorescent label to monitor the ability of a test compoundto modulate T-bet binding to a T-bet target molecule in a complex.Determining the ability of the test compound to bind T-bet can beaccomplished, for example, by coupling the compound with a radioisotope,enzymatic or fluorescent label such that binding of the compound toT-bet can be determined by detecting the labeled T-bet compound in acomplex. For example, T-bet targets can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound to interact with T-bet without the labeling of any of theinteractants. For example, a microphysiometer can be used to detect theinteraction of a compound with T-bet without the labeling of either thecompound or the T-bet (McConnell, H. M. et al. (1992) Science257:1906-1912). As used herein, a “microphysiometer” (e.g., Cytosensor)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between a compound and T-bet.

In another embodiment, a different (i.e., non-T-bet) molecule acting ina pathway involving T-bet that acts upstream or downstream of T-bet canbe included in an indicator composition for use in a screening assay.Compounds identified in a screening assay employing such a moleculewould also be useful in modulating T-bet activity, albeit indirectly. Anexemplary molecule with which T-bet interacts includes the Tec kinases,e.g., ITK or rlk kinase.

The cells used in the instant assays can be eukaryotic or prokaryotic inorigin. For example, in one embodiment, the cell is a bacterial cell. Inanother embodiment, the cell is a fungal cell, e.g., a yeast cell. Inanother embodiment, the cell is a vertebrate cell, e.g., an avian or amammalian cell. In a preferred embodiment, the cell is a human cell.

The cells of the invention can express endogenous T-bet (or anotherpolypeptide in a signaling pathway involving T-bet) or may be engineeredto do so. A cell that has been engineered to express the T-betpolypeptide or a non T-bet polypeptide which acts upstream or downstreamof T-bet can be produced by introducing into the cell an expressionvector encoding the T-bet polypeptide or a non T-bet polypeptide whichacts upstream or downstream of T-bet.

Recombinant expression vectors that can be used for expression of T-betpolypeptide or a non T-bet polypeptide which acts upstream or downstreamof T-bet in the indicator cell are known in the art. In one embodiment,within the expression vector the T-bet-coding sequences are operativelylinked to regulatory sequences that allow for inducible or constitutiveexpression of T-bet in the indicator cell (e.g., viral regulatorysequences, such as a cytomegalovirus promoter/enhancer, can be used).Use of a recombinant expression vector that allows for inducible orconstitutive expression of T-bet in the indicator cell is preferred foridentification of compounds that enhance or inhibit the activity ofT-bet. In an alternative embodiment, within the expression vector theT-bet-coding sequences are operatively linked to regulatory sequences ofthe endogenous T-bet gene (i.e., the promoter regulatory region derivedfrom the endogenous T-bet gene). Use of a recombinant expression vectorin which T-bet expression is controlled by the endogenous regulatorysequences is preferred for identification of compounds that enhance orinhibit the transcriptional expression of T-bet.

In methods in which a Th1-associated cytokine gene is utilized (e.g., asa reporter gene or as a readout to assess T-bet activity), preferably,the Th1-associated cytokine is interferon-γ or IL-2. As described in theappended examples, T-bet was isolated in a yeast one hybrid screeningassay based on its ability to bind to the IL-2 promoter. Accordingly, inone embodiment, a method of the invention utilizes a reporter geneconstruct containing this region of the proximal IL-2 promoter, mostpreferably nucleotides-240 to -220 of the IL-2 promoter. Other sequencesthat can be employed include: the consensus T-box site, the human IL-2promoter, the murine IL-2 promoter, the human IFN-γ intron III, twobinding sites in the murine IFN-γ proximal promoter. (Szabo et al. 2000.Cell 100:655-669).

In one embodiment, an inducible system can be constructed and used inhigh throughput cell-based screens to identify and characterize targetcompounds that affect the expression and/or activity of T-bet. Theinducible system can be constructed using a cell line that does notnormally produce IFN-γ, for example, by using a subclone of the adherent293T human embryonic kidney cell line that expresses the ecdysonereceptor, co-transfected with an ecdysone-driven T-bet expressionplasmid, and an IFN-γ promoter luciferase reporter. (Wakita t al. 2001.Biotechniques 31:414; No et al. Proceedings of the National Academy ofSciences USA 93:3346; Graham. 2002 Expert Opin. Biol. Ther. 2:525). Upontreatment with the insect hormone ecdysone, T-bet is expressed, theIFN-γ reporter is activated and luciferase activity is generated. Inthis system, T-bet confers on the cell line the ability to produceendogenous IFN-γ.

B. Cell-Free Assays

In another embodiment, the indicator composition is a cell freecomposition. T-bet or a non-T-bet polypeptide which acts upstream ordownstream of T-bet in a pathway involving T-bet expressed byrecombinant methods in a host cells or culture medium can be isolatedfrom the host cells, or cell culture medium using standard methods forpurifying polypeptides, for example, by ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for T-bet toproduce protein that can be used in a cell free composition.Alternatively, an extract of T-bet or non-T-bet expressing cells can beprepared for use as cell-free composition.

In one embodiment, compounds that specifically modulate T-bet activityare identified based on their ability to modulate the interaction ofT-bet with a target molecule to which T-bet binds. The target moleculecan be a DNA molecule, e.g., a T-bet-responsive element, such as theregulatory region of a cytokine gene) or a polypeptide molecule, e.g., aTec kinase. Suitable assays are known in the art that allow for thedetection of protein-protein interactions (e.g., immunoprecipitations,fluorescent polarization or energy transfer, two-hybrid assays and thelike) or that allow for the detection of interactions between a DNAbinding protein with a target DNA sequence (e.g., electrophoreticmobility shift assays, DNAse I footprinting assays and the like). Byperforming such assays in the presence and absence of test compounds,these assays can be used to identify compounds that modulate (e.g.,inhibit or enhance) the interaction of T-bet with a target molecule.

In one embodiment, the amount of binding of T-bet to the target moleculein the presence of the test compound is greater than the amount ofbinding of T-bet to the target molecule in the absence of the testcompound, in which case the test compound is identified as a compoundthat enhances binding of T-bet. In another embodiment, the amount ofbinding of the T-bet to the target molecule in the presence of the testcompound is less than the amount of binding of the T-bet to the targetmolecule in the absence of the test compound, in which case the testcompound is identified as a compound that inhibits binding of T-bet.

Binding of the test compound to the T-bet polypeptide can be determinedeither directly or indirectly as described above. Determining theability of the T-bet polypeptide to bind to a test compound can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705). As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In the methods of the invention for identifying test compounds thatmodulate an interaction between T-bet polypeptide and a target molecule,the full-length T-bet polypeptide may be used in the method, or,alternatively, only portions of the T-bet may be used. The degree ofinteraction between T-bet polypeptides and the target molecule can bedetermined, for example, by labeling one of the polypeptides with adetectable substance (e.g., a radiolabel), isolating the non-labeledpolypeptide and quantitating the amount of detectable substance that hasbecome associated with the non-labeled polypeptide. The assay can beused to identify test compounds that either stimulate or inhibit theinteraction between the T-bet protein and a target molecule. A testcompound that stimulates the interaction between the T-bet polypeptideand a target molecule is identified based upon its ability to increasethe degree of interaction between the T-bet polypeptide and a targetmolecule as compared to the degree of interaction in the absence of thetest compound. A test compound that inhibits the interaction between theT-bet polypeptide and a target molecule is identified based upon itsability to decrease the degree of interaction between the T-betpolypeptide and a target molecule as compared to the degree ofinteraction in the absence of the compound.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either T-bet or a T-bettarget molecule, a kinase, for example, to facilitate separation ofcomplexed from uncomplexed forms of one or both of the polypeptides, orto accommodate automation of the assay. Binding of a test compound to aT-bet polypeptide, or interaction of a T-bet polypeptide with a T-bettarget molecule in the presence and absence of a test compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the polypeptidesto be bound to a matrix. For example, glutathione-S-transferase/T-betfusion proteins or glutathione-S-transferase/target fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target polypeptide or T-bet polypeptide, and the mixtureincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads or microtitre plate wells are washed to remove any unboundcomponents, the matrix is immobilized in the case of beads, and complexformation is determined either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of T-bet binding or activity determined usingstandard techniques.

Other techniques for immobilizing polypeptides on matrices can also beused in the screening assays of the invention. For example, either aT-bet polypeptide or a T-bet target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated T-betpolypeptide or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies which are reactive with T-bet polypeptide ortarget molecules but which do not interfere with binding of the T-betpolypeptide to its target molecule can be derivatized to the wells ofthe plate, and unbound target or T-bet polypeptide is trapped in thewells by antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theT-bet polypeptide or target molecule, as well as enzyme-linked assayswhich rely on detecting an enzymatic activity associated with the T-betpolypeptide or target molecule.

In yet another aspect of the invention, the T-bet polypeptide orfragments thereof can be used as “bait proteins” in a two-hybrid assayor three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identifyother polypeptides, which bind to or interact with T-bet (“T-bet-bindingproteins” or “T-bet”) and are involved in T-bet activity. SuchT-bet-binding proteins are also likely to be involved in the propagationof signals by the T-bet polypeptides or T-bet targets as, for example,downstream elements of a T-bet-mediated signaling pathway.Alternatively, such T-bet-binding polypeptides are likely to be T-betinhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a T-betpolypeptide is fused to a gene encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aT-bet-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes thepolypeptide which interacts with the T-bet polypeptide.

In another embodiment, representational difference analysis (RDA) andmicrochip DNA array analysis to isolate T-bet target genes. For example,differential display or subtraction methods coupled with PCR (RDA; seee.g., Hubank, M. & Schatz, D. G. 1994. Nuc. Acid Res. 22, 5640-5648;Chang, Y., et al. 1994. Science 266, 1865; von Stein, O. D., et al.1997. Nuc. Acid Res. 25, 2598; Lisitsyn, N. & Wigler, M. 1993. Science259, 946) employing subtracted or unsubtracted probes or, most recently,DNA microchip array hybridization (Welford et al. 1998. Nucl. Acids.Res. 15:3059) can be used. In performing such assays, a variety of cellscan be used, e.g., normal cells, cells engineered to express T-bet, orcells from mice lacking T-bet or overexpressing T-bet (e.g., from atransgenic non-human animal) can be used.

In yet another embodiment, protcomic approaches to describe T-bet targetproteins can be performed. For example, subtractive analysis, analysisof expression patterns, identification of genotypic variations at theprotein level and protein identification and detection ofpost-translational modifications can be performed as described in, e.g.,Wang et al. (2002) J. Chromatogr. B. Technol. Biomed Life Sci. 782(1-2):291-306; Lubman et al. (2002) J. Chromatogr. B. Technol. Biomed LifeSci. 782(1-2): 183-96; and Rai et al. (2002) Arch. Pathol. Lab. Med.126(12):1518-26.

C. Assays Using T-Bet Deficient Cells

In another embodiment, the invention provides methods for identifyingcompounds that modulate a biological effect of T-bet using cellsdeficient in T-bet. As described in the Examples, inhibition of T-betactivity (e.g., by disruption of the T-bet gene) in B cells results in adeficiency of IgG2a production. Thus, cells deficient in T-bet can beused identify agents that modulate a biological response regulated byT-bet by means other than modulating T-bet itself (i.e., compounds that“rescue” the T-bet deficient phenotype). Alternatively, a “conditionalknock-out” system, in which the T-bet gene is rendered non-functional ina conditional manner, can be used to create T-bet deficient cells foruse in screening assays. For example, a tetracycline-regulated systemfor conditional disruption of a gene as described in WO 94/29442 andU.S. Pat. No. 5,650,298 can be used to create cells, or T-bet deficientanimals from which cells can be isolated, that can be rendered T-betdeficient in a controlled manner through modulation of the tetracyclineconcentration in contact with the cells. For assays relating to otherbiological effects of T-bet a similar conditional disruption approachcan be used or, alternatively, the RAG-2 deficient blastocystcomplementation system can be used to generate mice with lymphoid organsthat arise from embryonic stem cells with homozygous mutations of theT-bet gene. T-bet deficient lymphoid cells (e.g., thymic, splenic and/orlymph node cells) or purified T-bet deficient B cells from such animalscan be used in screening assays.

In the screening method, cells deficient in T-bet are contacted with atest compound and a biological response regulated by T-bet is monitored.Modulation of the response in T-bet deficient cells (as compared to anappropriate control such as, for example, untreated cells or cellstreated with a control agent) identifies a test compound as a modulatorof the T-bet regulated response.

In one embodiment, the test compound is administered directly to anon-human T-bet deficient animal, preferably a mouse (e.g., a mouse inwhich the T-bet gene is conditionally disrupted by means describedabove, or a chimeric mouse in which the lymphoid organs are deficient inT-bet as described above), to identify a test compound that modulatesthe in vivo responses of cells deficient in T-bet. In anotherembodiment, cells deficient in T-bet are isolated from the non-humanT-bet deficient animal, and contacted with the test compound ex vivo toidentify a test compound that modulates a response regulated by T-bet inthe cells deficient in T-bet.

Cells deficient in T-bet can be obtained from a non-human animalscreated to be deficient in T-bet. Preferred non-human animals includemonkeys, dogs, cats, mice, rats, cows, horses, goats and sheep. Inpreferred embodiments, the T-bet deficient animal is a mouse. Micedeficient in T-bet can be made as described in the Examples. Non-humananimals deficient in a particular gene product typically are created byhomologous recombination. Briefly, a vector is prepared which containsat least a portion of the T-bet gene into which a deletion, addition orsubstitution has been introduced to thereby alter, e.g., functionallydisrupt, the endogenous T-bet gene. The T-bet gene preferably is a mouseT-bet gene. For example, a mouse T-bet gene can be isolated from a mousegenomic DNA library using the mouse T-bet cDNA as a probe. The mouseT-bet gene then can be used to construct a homologous recombinationvector suitable for altering an endogenous T-bet gene in the mousegenome. In a preferred embodiment, the vector is designed such that,upon homologous recombination, the endogenous T-bet gene is functionallydisrupted (i.e., no longer encodes a functional polypeptide; alsoreferred to as a “knock out” vector). Alternatively, the vector can bedesigned such that, upon homologous recombination, the endogenous T-betgene is mutated or otherwise altered but still encodes functionalpolypeptide (e.g., the upstream regulatory region can be altered tothereby alter the expression of the endogenous T-bet polypeptide). Inthe homologous recombination vector, the altered portion of the T-betgene is flanked at its 5′ and 3′ ends by additional nucleic acid of theT-bet gene to allow for homologous recombination to occur between theexogenous T-bet gene carried by the vector and an endogenous T-bet genein an embryonic stem cell. The additional flanking T-bet nucleic acid isof sufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see e.g., Thomas, K. R.and Capecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedT-bet gene has homologously recombined with the endogenous T-bet geneare selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec etal.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; andWO 93/04169 by Berns et al.

In another embodiment, retroviral transduction of donor bone marrowcells from both wild type and T-bet null mice can be performed with theDN or dominant negative constructs to reconstitute irradiated RAGrecipients. This will result in the production of mice whose lymphoidcells express only a dominant negative version of T-bet. B cells fromthese mice can then be tested for compounds that modulate a biologicalresponse regulated by T-bet.

In one embodiment of the screening assay, compounds tested for theirability to modulate a biological response regulated by T-bet arecontacted with T-bet deficient cells by administering the test compoundto a non-human T-bet deficient animal in vivo and evaluating the effectof the test compound on the response in the animal. The test compoundcan be administered to a non-human T-bet deficient animal as apharmaceutical composition. Such compositions typically comprise thetest compound and a pharmaceutically acceptable carrier. As used hereinthe term “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal compounds, isotonic and absorption delaying compounds, andthe like, compatible with pharmaceutical administration. The use of suchmedia and compounds for pharmaceutically active substances is well knownin the art. Except insofar as any conventional media or compound isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

D. Test Compounds

A variety of test compounds can be evaluated using the screening assaysdescribed herein. In certain embodiments, the compounds to be tested canbe derived from libraries (i.e., are members of a library of compounds).While the use of libraries of peptides is well established in the art,new techniques have been developed which have allowed the production ofmixtures of other compounds, such as benzodiazepines (Bunin et al.(1992). J. Am. Chem. Soc. 114:10987; DeWitt et al. (1993). Proc. Natl.Acad. Sci. USA 90:6909) peptoids (Zuckermann. (1994). J. Med. Chem.37:2678) oligocarbamates (Cho et al. (1993). Science. 261:1303-), andhydantoins (DeWitt et al. supra). An approach for the synthesis ofmolecular libraries of small organic molecules with a diversity of104-105 as been described (Carell et al. (1994). Angew. Chem. Int. Ed.Engl. 33:2059-; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061-).

The compounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries, synthetic library methods requiringdeconvolution, the ‘one-bead one-compound’ library method, and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer CompoundDes. 12:145). Other exemplary methods for the synthesis of molecularlibraries can be found in the art, for example in: Erb et al. (1994).Proc. Natl. Acad. Sci. USA 91:11422-; Horwell et al. (1996)Immunopharmacology 33:68-; and in Gallop et al. (1994); J. Med. Chem.37:1233-.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); In stillanother embodiment, the combinatorial polypeptides are produced from acDNA library.

Exemplary compounds which can be screened for activity include, but arenot limited to, peptides, nucleic acids, carbohydrates, small organicmolecules, and natural product extract libraries.

Candidate/test compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam, K. S. et al. (1991) Nature354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang, Z. et al. (1993) Cell 72:767-778); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); 4) smallorganic and inorganic molecules (e.g., molecules obtained fromcombinatorial and natural product libraries); 5) enzymes (e.g.,endoribonucleases, hydrolases, nucleases, proteases, synthatases,isomerases, polymerases, kinases, phosphatases, oxido-reductases andATPases), and 6) mutant forms or T-bet molecules, e.g., dominantnegative mutant forms of the molecules.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) AnticancerCompound Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or phage (Scottand Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406;Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991)J. Mol. Biol. 222:301-310; Ladner supra.).

Compounds identified in the subject screening assays can be used inmethods of modulating one or more of the biological responses regulatedby T-bet. It will be understood that it may be desirable to formulatesuch compound(s) as pharmaceutical compositions (described supra) priorto contacting them with cells.

Once a test compound is identified that directly or indirectly modulatesT-bet expression and/or activity, by one of the variety of methodsdescribed hereinbefore, the selected test compound (or “compound ofinterest”) can then be further evaluated for its effect on cells, forexample by contacting the compound of interest with cells either in vivo(e.g., by administering the compound of interest to a subject) or exvivo (e.g., by isolating cells from the subject and contacting theisolated cells with the compound of interest or, alternatively, bycontacting the compound of interest with a cell line) and determiningthe effect of the compound of interest on the cells, as compared to anappropriate control (such as untreated cells or cells treated with acontrol compound, or carrier, that does not modulate the biologicalresponse). Compounds of interest can also be identified using structurebased drug design using techniques known in the art.

The instant invention also pertains to compounds identified in the aboveassays.

Methods for Modulating Biological Responses Regulated by T-Bet

Yet another aspect of the invention pertains to methods of modulatingT-bet expression and/or activity in a cell. The modulatory methods ofthe invention involve contacting the cell with an agent that modulatesT-bet expression and/or activity such that T-bet expression and/oractivity in the cell is modulated. In order for T-bet expression and/oractivity to be modulated in a cell, the cell is contacted with amodulatory agent in an amount sufficient to modulate the expressionand/or activity of T-bet.

In one embodiment, the modulatory methods of the invention are performedin vitro. In another embodiment, the modulatory methods of the inventionare performed in vivo, e.g., in a subject having a disorder or conditionthat would benefit from modulation of T-bet expression and/or activity.

The agent may act by modulating the activity of T-bet polypeptide in thecell, (e.g., by contacting a cell with an agent that, e.g., interferswith the binding of T-bet to a molecule with which it interacts, changesthe binding specificity of T-bet, or post-translationally modifiesT-bet) or the expression of T-bet, (e.g., by modulating transcription ofthe T-bet gene or translation of the T-bet mRNA).

Accordingly, the invention features methods for modulating one or morebiological responses regulated by T-bet by contacting the cells with amodulator of T-bet expression and/or activity such that the biologicalresponse is modulated.

As described in the appended Examples, T-bet has a variety of biologicaleffects on cells, including modulating amount of T helper-type 2 and/orT helper-type 1 cytokines produced by a cell, modulation of T celllineage commitment, modulating TGF-β mediated signaling, modulatingsignaling via the Jak1/STAT-1 pathway, modulating IgG class switching,and modulating B lymphocyte function.

In another embodiment, a gene whose transcription is modulated by T-betcan be modulated using the methods of the invention. Exemplary geneswhose expression is modulated by T-bet include, e.g., IFN-γ, IL-2, IL-4,IL-10, and TGF-β. in another embodiment, a biological response regulatedby T-bet can be modulated indirectly by modulating a non-T-bet moleculethat acts upstream or downstream of T-bet in a signal transductionpathway involving T-bet. For example, as demonstrated in the instantexamples, extracellular influences that modulate T-bet expression and/oractivity include TGF-β and IFN-γ. Therefore, agents that modulate TGF-βor IFN-γ or that modulate molecules in a TGF-β or IFN-γ signaltransduction pathway can be used to modulate T-bet.

The subject methods employ agents that modulate T-bet expression,processing, post-translational modification, or activity (or theexpression, processing, post-translational modification, or activity ofanother molecule in a T-bet signaling pathway) such that T-bet ismodulated. The subject methods are useful in both clinical andnon-clinical settings.

In one embodiment, the instant methods can be performed in vitro. Forexample, the production of a commercially valuable polypeptide, e.g., arecombinantly expressed polypeptide, can be increased by stimulating theIFN-γ pathway. In a preferred embodiment, T-bet can be modulated in acell in vitro and then the treated cells can be administered to asubject.

The subject invention can also be used to treat various conditions ordisorders that would benefit from modulation of T-bet. Exemplarydisorders that would benefit from modulation of T-bet expression and/oractivity are set forth herein. In one embodiment, the invention providesfor modulation of T-bet in vivo, by administering to the subject atherapeutically effective amount of a modulator of T-bet such that abiological effect of T-bet in a subject is modulated. For example, T-betcan be modulated to treat an autoimmune disorder, or animmunodeficiency.

The term “subject” is intended to include living organisms in which animmune response can be elicited. Preferred subjects are mammals.Particularly preferred subjects are humans. Other examples of subjectsinclude monkeys, dogs, cats, mice, rats cows, horses, goats, sheep aswell as other farm and companion animals. Modulation of T-bet expressionand/or activity, in humans as well as veterinary applications, providesa means to regulate disorders arising from aberrant T-bet expressionand/or activity in various disease states and is encompassed by thepresent invention.

Identification of compounds that modulate the biological effects ofT-bet by directly or indirectly modulating T-bet expression and/oractivity allows for selective manipulation of these biological effectsin a variety of clinical situations using the modulatory methods of theinvention. For example, the stimulatory methods of the invention (i.e.,methods that use a stimulatory agent) can result in increased expressionand/or activity of T-bet, which stimulates, e.g., IFN-γ production, IgGclass switching and the production of CD8 effector cells and whichinhibits, e.g., TGF-β, IL-2, IL-4, and IL-10, and can reduce tolerance(e.g., by reducing the number or percentage of Tr cells). In contrast,the inhibitory methods of the invention (i.e., methods that use an agentthat inhibits T-bet) can have the opposite effects.

Application of the modulatory methods of the invention to the treatmentof a disorder may result in cure of the disorder, a decrease in the typeor number of symptoms associated with the disorder, either in the longterm or short term (i.e., amelioration of the condition) or simply atransient beneficial effect to the subject.

Application of the immunomodulatory methods of the invention isdescribed in further detail below.

A. Inhibitory Agents

According to a modulatory method of the invention, T-bet expressionand/or activity is inhibited in a cell by contacting the cell with aninhibitory agent. Inhibitory agents of the invention can be, forexample, intracellular binding molecules that act to inhibit theexpression and/or activity of T-bet. As used herein, the term“intracellular binding molecule” is intended to include molecules thatact intracellularly to inhibit the expression and/or activity of apolypeptide by binding to the polypeptide itself, to a nucleic acid(e.g., an mRNA molecule) that encodes the polypeptide or to a targetwith which the polypeptide normally interacts (e.g., to a DNA targetsequence to which T-bet binds). Examples of intracellular bindingmolecules, described in further detail below, include antisense T-betnucleic acid molecules (e.g., to inhibit translation of T-bet mRNA),intracellular anti-T-bet antibodies (e.g., to inhibit the activity ofT-bet polypeptide) and dominant negative mutants of the T-betpolypeptide.

In one embodiment, an inhibitory agent of the invention is an antisensenucleic acid molecule that is complementary to a gene encoding T-bet orto a portion of said gene, or a recombinant expression vector encodingsaid antisense nucleic acid molecule. The use of antisense nucleic acidsto downregulate the expression of a particular polypeptide in a cell iswell known in the art (see e.g., Weintraub, H. et al., Antisense RNA asa molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol.1(1) 1986; Askari, F. K. and McDonnell, W. M. (1996) N. Eng. J. Med.334:316-318; Bennett, M. R. and Schwartz, S. M. (1995) Circulation92:1981-1993; Mercola, D. and Cohen, J. S. (1995) Cancer Gene Ther.2:47-59; Rossi, J. J. (1995) Br. Med. Bull. 51:217-225; Wagner, R. W.(1994) Nature 372:333-335). An antisense nucleic acid molecule comprisesa nucleotide sequence that is complementary to the coding strand ofanother nucleic acid molecule (e.g., an mRNA sequence) and accordinglyis capable of hydrogen bonding to the coding strand of the other nucleicacid molecule. Antisense sequences complementary to a sequence of anmRNA can be complementary to a sequence found in the coding region ofthe mRNA, the 5′ or 3′ untranslated region of the mRNA or a regionbridging the coding region and an untranslated region (e.g., at thejunction of the 5′ untranslated region and the coding region).Furthermore, an antisense nucleic acid can be complementary in sequenceto a regulatory region of the gene encoding the mRNA, for instance atranscription initiation sequence or regulatory element. Preferably, anantisense nucleic acid is designed so as to be complementary to a regionpreceding or spanning the initiation codon on the coding strand or inthe 3′ untranslated region of an mRNA. An antisense nucleic acid forinhibiting the expression of T-bet polypeptide in a cell can be designedbased upon the nucleotide sequence encoding the T-bet polypeptide (e.g.,SEQ ID NO: 1 or 3), constructed according to the rules of Watson andCrick base pairing.

An antisense nucleic acid can exist in a variety of different forms. Forexample, the antisense nucleic acid can be an oligonucleotide that iscomplementary to only a portion of a T-bet gene. An antisenseoligonucleotides can be constructed using chemical synthesis proceduresknown in the art. An antisense oligonucleotide can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g. phosphorothioatederivatives and acridine substituted nucleotides can be used. To inhibitT-bet expression in cells in culture, one or more antisenseoligonucleotides can be added to cells in culture media, typically atabout 200 μg oligonucleotide/ml.

Alternatively, an antisense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i.e., nucleic acid transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Regulatory sequences operatively linked to anucleic acid cloned in the antisense orientation can be chosen whichdirect the expression of the antisense RNA molecule in a cell ofinterest, for instance promoters and/or enhancers or other regulatorysequences can be chosen which direct constitutive, tissue specific orinducible expression of antisense RNA. For example, for inducibleexpression of antisense RNA, an inducible eukaryotic regulatory system,such as the Tet system (e.g., as described in Gossen, M. and Bujard, H.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995)Science 268:1766-1769; PCT Publication No. WO 94/29442; and PCTPublication No. WO 96/01313) can be used. The antisense expressionvector is prepared as described above for recombinant expressionvectors, except that the cDNA (or portion thereof) is cloned into thevector in the antisense orientation. The antisense expression vector canbe in the form of, for example, a recombinant plasmid, phagemid orattenuated virus. The antisense expression vector is introduced intocells using a standard transfection technique, as described above forrecombinant expression vectors.

In another embodiment, an antisense nucleic acid for use as aninhibitory agent is a ribozyme. Ribozymes are catalytic RNA moleculeswith ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region (for reviews on ribozymes see e.g., Ohkawa, J. etal. (1995) J. Biochem. 118:251-258; Sigurdsson, S. T. and Eckstein, F.(1995) Trends Biotechnol. 13:286-289; Rossi, J. J. (1995) TrendsBiotechnol. 13:301-306; Kiehntopf, M. et al. (1995) J. Mol. Med.73:65-71). A ribozyme having specificity for T-bet mRNA can be designedbased upon the nucleotide sequence of the T-bet cDNA. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thebase sequence of the active site is complementary to the base sequenceto be cleaved in a T-bet mRNA. See for example U.S. Pat. Nos. 4,987,071and 5,116,742, both by Cech et al. Alternatively, T-bet mRNA can be usedto select a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See for example Bartel, D. and Szostak, J. W.(1993) Science 261: 1411-1418.

In another embodiment, RNAi can be used to inhibit T-bet expression. RNAinterference (RNAi is a post-transcriptional, targeted gene-silencingtechnique that uses double-stranded RNA (dsRNA) to degrade messenger RNA(mRNA) containing the same sequence as the dsRNA (Sharp, P. A. andZamore, P. D. 287, 2431-2432 (2000); Zamore, P. D., et al. Cell 101,25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197 (1999)). Theprocess occurs when an endogenous ribonuclease cleaves the longer dsRNAinto shorter, 21- or 22-nucleotide-long RNAs, termed small interferingRNAs or siRNAs. The smaller RNA segments then mediate the degradation ofthe target mRNA. Kits for synthesis of RNAi are commercially availablefrom, e.g. New England Biolabs and Ambion. In one embodiment one or moreof the chemistries described above for use in antisense RNA can beemployed.

Another type of inhibitory agent that can be used to inhibit theexpression and/or activity of T-bet in a cell is an intracellularantibody specific for the T-bet polypeptide. The use of intracellularantibodies to inhibit polypeptide function in a cell is known in the art(see e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca,S. et al. (1990) EMBO J. 9:101-108; Werge, T. M. et al. (1990) FEBSLetters 274:193-198; Carlson, J. R. (1993) Proc. Natl. Acad. Sci. USA90:7427-7428; Marasco, W. A. et al. (1993) Proc. Natl. Acad. Sci. USA90:7889-7893; Biocca, S. et al. (1994) Bio/Technology 12:396-399; Chen,S-Y. et al. (1994) Human Gene Therapy 5:595-601; Duan, L et al. (1994)Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S-Y. et al. (1994) Proc.Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al. (1994) J. Biol.Chem. 269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys.Res. Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

To inhibit polypeptide activity using an intracellular antibody, arecombinant expression vector is prepared which encodes the antibodychains in a form such that, upon introduction of the vector into a cell,the antibody chains are expressed as a functional antibody in anintracellular compartment of the cell. For inhibition of T-bet activityaccording to the inhibitory methods of the invention, an intracellularantibody that specifically binds the T-bet polypeptide is expressed inthe cytoplasm of the cell. To prepare an intracellular antibodyexpression vector, antibody light and heavy chain cDNAs encodingantibody chains specific for the target protein of interest, e.g.,T-bet, are isolated, typically from a hybridoma that secretes amonoclonal antibody specific for the T-bet polypeptide. Hybridomassecreting anti-T-bet monoclonal antibodies, or recombinant anti-T-betmonoclonal antibodies, can be prepared as described above. Monoclonalantibodies specific for T-bet polypeptide have been identified suchhybridoma-derived monoclonal antibodies can be used or a recombinantantibody from a combinatorial library can be used), DNAs encoding thelight and heavy chains of the monoclonal antibody are isolated bystandard molecular biology techniques. For hybridoma derived antibodies,light and heavy chain cDNAs can be obtained, for example, by PCRamplification or cDNA library screening. For recombinant antibodies,such as from a phage display library, cDNA encoding the light and heavychains can be recovered from the display package (e.g., phage) isolatedduring the library screening process. Nucleotide sequences of antibodylight and heavy chain genes from which PCR primers or cDNA libraryprobes can be prepared are known in the art. For example, many suchsequences are disclosed in Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242 and in the“Vbase” human germline sequence database.

Once obtained, the antibody light and heavy chain sequences are clonedinto a recombinant expression vector using standard methods. To allowfor cytoplasmic expression of the light and heavy chains, the nucleotidesequences encoding the hydrophobic leaders of the light and heavy chainsare removed. An intracellular antibody expression vector can encode anintracellular antibody in one of several different forms. For example,in one embodiment, the vector encodes full-length antibody light andheavy chains such that a full-length antibody is expressedintracellularly. In another embodiment, the vector encodes a full-lengthlight chain but only the VH/CH1 region of the heavy chain such that aFab fragment is expressed intracellularly. In the most preferredembodiment, the vector encodes a single chain antibody (scFv) whereinthe variable regions of the light and heavy chains are linked by aflexible peptide linker (e.g., (Gly4Ser)₃) (SEQ ID NO:16) and expressedas a single chain molecule. To inhibit T-bet activity in a cell, theexpression vector encoding the anti-T-bet intracellular antibody isintroduced into the cell by standard transfection methods, as discussedhereinbefore.

Yet another form of an inhibitory agent of the invention is aninhibitory form of T-bet, also referred to herein as a dominant negativeinhibitor, e.g., a form of T-bet in comprising engrailed sequences astaught in the instant examples. Such a molecule can be introduced into acell using standard techniques to allow for expression of the alteredform of T-bet, in the cell.

In a preferred embodiment, inhibitory compounds of the invention thatreduce the activity and/or expression of T-bet also reduce, for example,the onset or severity of autoimmune diseases such as MS, arthritis,Chrohn's disease and Th1 mediated experimental colitits. The inhibitorycompounds of the invention that reduce the activity and/or expression ofT-bet also increase, for example, the onset or severity of diseasesincluding, asthma and Th2 mediated colitits.

In another embodiment, an inhibitory agent of the invention is a smallmolecule which interacts with the T-bet protein to thereby inhibit theactivity of T-bet. Small molecule inhibitors of T-bet can be identifiedusing database searching programs capable of scanning a database ofsmall molecules of known three-dimensional structure for candidateswhich fit into the target protein site known in the art. Suitablesoftware programs include, for example, CATALYST (Molecular SimulationsInc., San Diego, Calif.), UNITY (Tripos Inc., St Louis, Mo.), FLEXX(Rarey et al., J. Mol. Biol. 261: 470-489 (1996)), CHEM-3DBS (OxfordMolecular Group, Oxford, UK), DOCK (Kuntz et al., J. Mol. Biol 161:269-288 (1982)), and MACCS-3D (MDL Information Systems Inc., SanLeandro, Calif.).

The molecules found in the search may not necessarily be leadsthemselves, however, such candidates might act as the framework forfurther design, providing molecular skeletons to which appropriateatomic replacements can be made. The scaffold, functional groups,linkers and/or monomers may be changed to maximize the electrostatic,hydrogen bonding, and hydrophobic interactions with the target protein.Goodford (Goodford J Med Chem 28:849-857 (1985)) has produced a computerprogram, GRID, which seeks to determine regions of high affinity fordifferent chemical groups (termed probes) on the molecular surface ofthe binding site. GRID hence provides a tool for suggestingmodifications to known ligands that might enhance binding. A range offactors, including electrostatic interactions, hydrogen bonding,hydrophobic interactions, desolvation effects, conformational strain ormobility, chelation and cooperative interaction and motions of ligandand enzyme, all influence the binding effect and should be taken intoaccount in attempts to design small molecule inhibitors.

Small molecule inhibitors of T-bet can also be identified usingcomputer-assisted molecular design methods comprising searching forfragments which fit into a binding region subsite and link to apredefined scaffold can be used. The scaffold itself may be identifiedin such a manner. Programs suitable for the searching of such functionalgroups and monomers include LUDI (Boehm. J Comp. Aid. Mol. Des. 6:61-78(1992)), CAVEAT (Bartlett et al. in “Molecular Recognition in Chemicaland Biological Problems”, special publication of The Royal Chem. Soc.,78:182-196 (1989)) and MCSS (Miranker et al. Proteins 11: 29-34 (1991)).

Yet another computer-assisted molecular design method for identifyingsmall molecule inhibitors of the T-bet protein comprises the de novosynthesis of potential inhibitors by algorithmic connection of smallmolecular fragments that will exhibit the desired structural andelectrostatic complementarity with the active binding site of the T-betprotein. The methodology employs a large template set of small moleculeswith are iteratively pieced together in a model of the T-bet bindingsite. Programs suitable for this task include GROW (Moon et al. Proteins11:314-328 (1991)) and SPROUT (Gillet et al. J Comp. Aid. Mol. Des.7:127 (1993)).

The suitability of small molecule inhibitor candidates can be determinedusing an empirical scoring function, which can rank the bindingaffinities for a set of inhibitors. For an example of such a method seeMuegge et al. and references therein (Muegge et al., J Med. Chem.42:791-804 (1999)). Other modeling techniques can be used in accordancewith this invention, for example, those described by Cohen et al. (J.Med. Chem. 33: 883-894 (1994)); Navia et al. (Current Opinions inStructural Biology 2: 202-210 (1992)); Baldwin et al. (J. Med. Chem. 32:2510-2513 (1989)); Appelt et al. (J. Med. Chem. 34: 1925-1934 (1991));and Ealick et al. (Proc. Nat. Acad. Sci. USA 88: 11540-11544 (1991)).

Other inhibitory agents that can be used to inhibit the expressionand/or activity of a T-bet polypeptide include chemical compounds thatdirectly inhibit T-bet or compounds that inhibit the interaction betweenT-bet and target DNA or another polypeptide. Such compounds can beidentified using screening assays that select for such compounds, asdescribed in detail above.

B. Stimulatory Agents

According to a modulatory method of the invention, T-bet expressionand/or activity is stimulated in a cell by contacting the cell with astimulatory agent. Examples of such stimulatory agents include activeT-bet polypeptide and nucleic acid molecules encoding T-bet that areintroduced into the cell to increase T-bet expression and/or activity inthe cell. A preferred stimulatory agent is a nucleic acid moleculeencoding a T-bet polypeptide, wherein the nucleic acid molecule isintroduced into the cell in a form suitable for expression of the activeT-bet polypeptide in the cell. To express a T-bet polypeptide in a cell,typically a T-bet-encoding DNA is first introduced into a recombinantexpression vector using standard molecular biology techniques, asdescribed herein. A T-bet-encoding DNA can be obtained, for example, byamplification using the polymerase chain reaction (PCR), using primersbased on the T-bet nucleotide sequence. Following isolation oramplification of T-bet-encoding DNA, the DNA fragment is introduced intoan expression vector and transfected into target cells by standardmethods, as described herein.

In one embodiment, stimulatory compounds of the invention include agentsthat increase the activity and/or expression of T-bet to therebydecrease, for example, the activation of the IL-2 promoter, activationof IL-4 production, activate the production and signaling of TGF-β andthe increase of the production of Tr cells. In another embodiment,stimulatory compounds of the invention include agents that increase theactivity and/or expression of T-bet to thereby increase, for example,activation of IFN-γ, stimulation of pathogenic autoantibody production,IgG class switching and production of CD8 effector cells.

In a preferred embodiment, stimulatory compounds of the invention thatincrease the activity and/or expression of T-bet also increase, forexample, the onset or severity of autoimmune diseases such as MS,arthritis, Crohn's disease and Th1 mediated experimental colitits. Thestimulatory compounds of the invention that increase the activity and/orexpression of T-bet also reduce, for example, the onset or severity ofdiseases including, asthma and Th2 mediated colitits.

Other stimulatory agents that can be used to stimulate the activity of aT-bet polypeptide are chemical compounds that stimulate T-bet activityin cells, such as compounds that directly stimulate T-bet polypeptideand compounds that promote the interaction between T-bet and target DNAor other polypeptides. Such compounds can be identified using screeningassays that select for such compounds, as described in detail above.

The modulatory methods of the invention can be performed in vitro (e.g.,by culturing the cell with the agent or by introducing the agent intocells in culture) or, alternatively, in vivo (e.g., by administering theagent to a subject or by introducing the agent into cells of a subject,such as by gene therapy). For practicing the modulatory method in vitro,cells can be obtained from a subject by standard methods and incubated(i.e., cultured) in vitro with a modulatory agent of the invention tomodulate T-bet expression and/or activity in the cells. For example,peripheral blood mononuclear cells (PBMCs) can be obtained from asubject and isolated by density gradient centrifugation, e.g., withFicoll/Hypaque. Specific cell populations can be depleted or enrichedusing standard methods. For example, T cells can be enriched forexample, by positive selection using antibodies to T cell surfacemarkers, for example by incubating cells with a specific primarymonoclonal antibody (mAb), followed by isolation of cells that bind themAb using magnetic beads coated with a secondary antibody that binds theprimary mAb. Specific cell populations can also be isolated byfluorescence activated cell sorting according to standard methods. Ifdesired, cells treated in vitro with a modulatory agent of the inventioncan be readministered to the subject. For administration to a subject,it may be preferable to first remove residual agents in the culture fromthe cells before administering them to the subject. This can be done forexample by a Ficoll/Hypaque gradient centrifugation of the cells. Forfurther discussion of ex vivo genetic modification of cells followed byreadministration to a subject, see also U.S. Pat. No. 5,399,346 by W. F.Anderson et al.

For stimulatory or inhibitory agents that comprise nucleic acids(including recombinant expression vectors encoding T-bet polypeptide,antisense RNA, intracellular antibodies or dominant negativeinhibitors), the agents can be introduced into cells of the subjectusing methods known in the art for introducing nucleic acid (e.g., DNA)into cells in vivo. Examples of such methods encompass both non-viraland viral methods, including:

Direct Injection: Naked DNA can be introduced into cells in vivo bydirectly injecting the DNA into the cells (see e.g., Acsadi et al.(1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468).For example, a delivery apparatus (e.g., a “gene gun”) for injecting DNAinto cells in vivo can be used. Such an apparatus is commerciallyavailable (e.g., from BioRad).

Cationic Lipids: Naked DNA can be introduced into cells in vivo bycomplexing the DNA with cationic lipids or encapsulating the DNA incationic liposomes. Examples of suitable cationic lipid formulationsinclude N-[-1-(2,3-dioleoyloxy)propyl]N,N,N-triethylammonium chloride(DOTMA) and a 1:1 molar ratio of1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE)and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan, J. J. etal. (1995) Gene Therapy 2:38-49; San, H. et al. (1993) Human GeneTherapy 4:781-788).

Receptor-Mediated DNA Uptake: Naked DNA can also be introduced intocells in vivo by complexing the DNA to a cation, such as polylysine,which is coupled to a ligand for a cell-surface receptor (see forexample Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson etal. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320).Binding of the DNA-ligand complex to the receptor facilitates uptake ofthe DNA by receptor-mediated endocytosis. A DNA-ligand complex linked toadenovirus capsids which naturally disrupt endosomes, thereby releasingmaterial into the cytoplasm can be used to avoid degradation of thecomplex by intracellular lysosomes (see for example Curiel et al. (1991)Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.Acad. Sci. USA 90:2122-2126).

Retroviruses: Defective retroviruses are well characterized for use ingene transfer for gene therapy purposes (for a review see Miller, A. D.(1990) Blood 76:271). A recombinant retrovirus can be constructed havinga nucleotide sequences of interest incorporated into the retroviralgenome. Additionally, portions of the retroviral genome can be removedto render the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions which can be used toinfect a target cell through the use of a helper virus by standardtechniques. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are well known to those skilled in the art.Examples of suitable packaging virus lines include ψ Crip, ψCre, ψ2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Retroviral vectors requiretarget cell division in order for the retroviral genome (and foreignnucleic acid inserted into it) to be integrated into the host genome tostably introduce nucleic acid into the cell. Thus, it may be necessaryto stimulate replication of the target cell.

Adenoviruses: The genome of an adenovirus can be manipulated such thatit encodes and expresses a gene product of interest but is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld etal. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses are advantageous in that they do not require dividing cellsto be effective gene delivery vehicles and can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin etal. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use are deletedfor all or parts of the viral E1 and E3 genes but retain as much as 80%of the adenoviral genetic material.

Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product.

In a preferred embodiment, a retroviral expression vector encoding T-betis used to express T-bet polypeptide in cells in vivo, to therebystimulate T-bet polypeptide activity in vivo. Such retroviral vectorscan be prepared according to standard methods known in the art(discussed further above).

A modulatory agent, such as a chemical compound, can be administered toa subject as a pharmaceutical composition. Such compositions typicallycomprise the modulatory agent and a pharmaceutically acceptable carrier.As used herein the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions. Pharmaceutical compositions can beprepared as described above in subsection IV.

The identification of T-bet as a key regulator of the development of Th1cells described herein, and in the repression of the Th2 phenotype,allows for selective manipulation of T cell subsets in a variety ofclinical situations using the modulatory methods of the invention. Thestimulatory methods of the invention (i.e., methods that use astimulatory agent to enhance T-bet expression and/or activity) result inproduction of IFN-γ, with concomitant promotion of a Th1 response anddownregulation of both IL-2 and IL-4, thus downmodulating the Th2response. In contrast, the inhibitory methods of the invention (i.e.,methods that use an inhibitory agent to downmodulate T-bet expressionand/or activity) inhibit the production of IFN-γ, with concomitantdownregulation of a Th1 response and promotion of a Th2 response. Thus,to treat a disease condition wherein a Th1 response is beneficial, astimulatory method of the invention is selected such that Th1 responsesare promoted while downregulating Th2 responses. Alternatively, to treata disease condition wherein a Th2 response is beneficial, an inhibitorymethod of the invention is selected such that Th1 responses aredownregulated while promoting Th2 responses. Application of the methodsof the invention to the treatment of diseases or conditions may resultin cure of the condition, a decrease in the type or number of symptomsassociated with the condition, either in the long term or short term(i.e., amelioration of the condition) or simply a transient beneficialeffect to the subject.

Numerous diseases or conditions associated with a predominant Th1 orTh2-type response have been identified and would benefit from modulationof the type of response mounted in the individual suffering from thedisease condition. Application of the immunomodulatory methods of theinvention to such diseases or conditions is described in further detailbelow.

A. Allergies

Allergies are mediated through IgE antibodies whose production isregulated by the activity of Th2 cells and the cytokines producedthereby. In allergic reactions, IL-4 is produced by Th2 cells, whichfurther stimulates production of IgE antibodies and activation of cellsthat mediate allergic reactions, i.e., mast cells and basophils. IL-4also plays an important role in eosinophil mediated inflammatoryreactions. Accordingly, the stimulatory methods of the invention can beused to inhibit the production of Th2-associated cytokines, and inparticular IL-4, in allergic patients as a means to downregulateproduction of pathogenic IgE antibodies. A stimulatory agent may bedirectly administered to the subject or cells (e.g., Thp cells or Th2cells) may be obtained from the subject, contacted with a stimulatoryagent ex vivo, and readministered to the subject. Moreover, in certainsituations it may be beneficial to coadminister to the subject theallergen together with the stimulatory agent or cells treated with thestimulatory agent to inhibit (e.g., desensitize) the allergen-specificresponse. The treatment may be further enhanced by administering otherTh1-promoting agents, such as the cytokine IL-12 or antibodies toTh2-associated cytokines (e.g., anti-IL-4 antibodies), to the allergicsubject in amounts sufficient to further stimulate a Th1-type response.

B. Cancer

The expression of Th2-promoting cytokines has been reported to beelevated in cancer patients (see e.g., Yamamura, M., et al (1993) J.Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad.Sci. USA 89:7708-7712) and malignant disease is often associated with ashift from Th1 type responses to Th2 type responses along with aworsening of the course of the disease. Accordingly, the stimulatorymethods of the invention can be used to inhibit the production ofTh2-associated cytokines in cancer patients, as a means to counteractthe Th1 to Th2 shift and thereby promote an ongoing Th1 response in thepatients to ameliorate the course of the disease. The stimulatory methodcan involve either direct administration of an stimulatory agent to asubject with cancer or ex vivo treatment of cells obtained from thesubject (e.g., Thp or Th2 cells) with a stimulatory agent followed byreadministration of the cells to the subject. The treatment may befurther enhanced by administering other Th1-promoting agents, such asthe cytokine IL-12 or antibodies to Th2-associated cytokines (e.g.,anti-IL-4 antibodies), to the recipient in amounts sufficient to furtherstimulate a Th1-type response.

C. Infectious Diseases

The expression of Th2-promoting cytokines also has been reported toincrease during a variety of infectious diseases, including HIVinfection, tuberculosis, leishmaniasis, schistosomiasis, filarialnematode infection and intestinal nematode infection (see e.g.; Shearer,G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, Mand Shearer, G. M. (1993) Immunology Today 14:107-111; Fauci, A. S.(1988) Science 239:617-623; Locksley, R. M. and Scott, P. (1992)Immunoparasitology Today 1:A58-A61; Pearce, E. J., et al. (1991) J. Exp.Med. 173:159-166; Grzych, J-M., et al. (1991) J. Immunol. 141:1322-1327;Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270; Bancroft, A.J., et al. (1993) J. Immunol. 150:1395-1402; Pearlman, E., et al. (1993)Infect. Immun. 61:1105-1112; Else, K. J., et al. (1994) J. Exp. Med.179:347-351) and such infectious diseases are also associated with a Th1to Th2 shift in the immune response. Accordingly, the stimulatorymethods of the invention can be used to inhibit the production ofTh2-associated cytokines in subjects with infectious diseases, as ameans to counteract the Th1 to Th2 shift and thereby promote an ongoingTh1 response in the patients to ameliorate the course of the infection.The stimulatory method can involve either direct administration of aninhibitory agent to a subject with an infectious disease or ex vivotreatment of cells obtained from the subject (e.g., Thp or Th2 cells)with a stimulatory agent followed by readministration of the cells tothe subject. The treatment may be further enhanced by administeringother Th1-promoting agents, such as the cytokine IL-12 or antibodies toTh2-associated cytokines (e.g., anti-IL-4 antibodies), to the recipientin amounts sufficient to further stimulate a Th1-type response.

D. Autoimmune Diseases

The inhibitory methods of the invention can be used therapeutically inthe treatment of autoimmune diseases that are associated with a Th2-typedysfunction. Many autoimmune disorders are the result of inappropriateactivation of T cells that are reactive against self tissue and thatpromote the production of cytokines and autoantibodies involved in thepathology of the diseases. Modulation of T helper-type responses canhave an effect on the course of the autoimmune disease. For example, inexperimental allergic encephalomyelitis (EAE), stimulation of a Th2-typeresponse by administration of IL-4 at the time of the induction of thedisease diminishes the intensity of the autoimmune disease (Paul, W. E.,et al. (1994) Cell 76:241-251). Furthermore, recovery of the animalsfrom the disease has been shown to be associated with an increase in aTh2-type response as evidenced by an increase of Th2-specific cytokines(Koury, S. J., et al. (1992) J. Exp. Med. 176:1355-1364). Moreover, Tcells that can suppress EAE secrete Th2-specific cytokines (Chen, C., etal. (1994) Immunity 1:147-154). Since stimulation of a Th2-type responsein EAE has a protective effect against the disease, stimulation of a Th2response in subjects with multiple sclerosis (for which EAE is a model)is likely to be beneficial therapeutically. The inhibitory methods ofthe invention can be used to effect such a decrease.

Similarly, stimulation of a Th2-type response in type I diabetes in miceprovides a protective effect against the disease. Indeed, treatment ofNOD mice with IL-4 (which promotes a Th2 response) prevents or delaysonset of type I diabetes that normally develops in these mice (Rapoport,M. J., et al. (1993) J. Exp. Med. 178:87-99). Thus, stimulation of a Th2response in a subject suffering from or susceptible to diabetes mayameliorate the effects of the disease or inhibit the onset of thedisease.

Yet another autoimmune disease in which stimulation of a Th2-typeresponse may be beneficial is rheumatoid arthritis (RA). Studies haveshown that patients with rheumatoid arthritis have predominantly Th1cells in synovial tissue (Simon, A. K., et al. (1994) Proc. Natl. Acad.Sci. USA 91:8562-8566). By stimulating a Th2 response in a subject withRA, the detrimental Th1 response can be concomitantly downmodulated tothereby ameliorate the effects of the disease.

Accordingly, the inhibitory methods of the invention can be used tostimulate production of Th2-associated cytokines in subjects sufferingfrom, or susceptible to, an autoimmune disease in which a Th2-typeresponse is beneficial to the course of the disease. The inhibitorymethod can involve either direct administration of an inhibitory agentto the subject or ex vivo treatment of cells obtained from the subject(e.g., Thp, Th1 cells, B cells, non-lymphoid cells) with an inhibitoryagent followed by readministration of the cells to the subject. Thetreatment may be further enhanced by administering other Th2-promotingagents, such as IL-4 itself or antibodies to Th1-associated cytokines,to the subject in amounts sufficient to further stimulate a Th2-typeresponse.

In contrast to the autoimmune diseases described above in which a Th2response is desirable, other autoimmune diseases may be ameliorated by aTh1-type response. Such diseases can be treated using a stimulatoryagent of the invention (as described above for cancer and infectiousdiseases). The treatment may be further enhanced by administrating aTh1-promoting cytokine (e.g., IFN-γ) to the subject in amountssufficient to further stimulate a Th1-type response.

The efficacy of agents for treating autoimmune diseases can be tested inthe above described animal models of human diseases (e.g., EAE as amodel of multiple sclerosis and the NOD mice as a model for diabetes) orother well characterized animal models of human autoimmune diseases.Such animal models include the mrl/lpr/lpr mouse as a model for lupuserythematosus, murine collagen-induced arthritis as a model forrheumatoid arthritis, and murine experimental myasthenia gravis (seePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.840-856). A modulatory (i.e., stimulatory or inhibitory) agent of theinvention is administered to test animals and the course of the diseasein the test animals is then monitored by the standard methods for theparticular model being used. Effectiveness of the modulatory agent isevidenced by amelioration of the disease condition in animals treatedwith the agent as compared to untreated animals (or animals treated witha control agent).

Non-limiting examples of autoimmune diseases, disorders and conditionshaving an autoimmune component that may be treated according to theinvention include diabetes mellitus, arthritis (including rheumatoidarthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriaticarthritis), multiple sclerosis, myasthenia gravis, systemic lupuserythematosis, autoimmune thyroiditis, dermatitis (including atopicdermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome,including keratoconjunctivitis sicca secondary to Sjögren's Syndrome,alopecia areata, allergic responses due to arthropod bite reactions,Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, allergic asthma, cutaneouslupus erythematosus, scleroderma, vaginitis, proctitis, compounderuptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, and interstitial lungfibrosis.

In a particular embodiment, diseases, disorders and conditions that maybe treated by the methods of the invention include Crohn's disease andulcerative colitis, which are the two major forms of inflammatory boweldiseases (IBD) in humans. Cytokines produced by T lymphocytes appear toinitiate and perpetuate chronic intestinal inflammation. Crohn's diseaseis associated with increased production of T helper 1 (Th1) typecytokines such as IFN-γ and TNF. Ulcerative colitis is generallyassociated with T cells producing large amounts of the Th2 typecytokines and is referred to herein as “Th2-mediated colitis.”“Th1-mediated colitis” refers to a Crohn's disease profile as well as tothe Th1 type response which can occur in ulcerative colitis. InTh1-mediated colitis, agents which inhibit the activity of T-bet providea protective effect. In Th2-mediated colitis, agents which stimulate theactivity of T-bet provide a protective effect.

In another particular embodiment, diseases, disorders and conditionsthat may be treated by the methods of the invention include asthma,which is a disease of the bronchial tubes, or airways of the lungs,characterized by tightening of these airways. Production of IL-4, IL-5and IL-13 has been associated with the development of an asthma-likephenotype. Accordingly, agents of the invention which stimulate theactivity of T-bet provide a protective effect against asthma.

E. Transplantation

While graft rejection or graft acceptance may not be attributableexclusively to the action of a particular T cell subset (i.e., Th1 orTh2 cells) in the graft recipient (for a discussion see Dallman, M. J.(1995) Curr. Opin. Immunol. 7:632-638), numerous studies have implicateda predominant Th2 response in prolonged graft survival or a predominantTh1 response in graft rejection. For example, graft acceptance has beenassociated with production of a Th2 cytokine pattern and/or graftrejection has been associated with production of a Th1 cytokine pattern(see e.g., Takeuchi, T. et al. (1992) Transplantation 53:1281-1291;Tzakis, A. G. et al. (1994) J. Pediatr. Surg. 29:754-756; Thai, N. L. etal. (1995) Transplantation 59:274-281). Additionally, adoptive transferof cells having a Th2 cytokine phenotype prolongs skin graft survival(Maeda, H. et al. (1994) Int. Immunol. 6:855-862) and reducesgraft-versus-host disease (Fowler, D. H. et al. (1994) Blood84:3540-3549; Fowler, D. H. et al. (1994) Prog. Clin. Biol. Res.389:533-540). Still further, administration of IL-4, which promotes Th2differentiation, prolongs cardiac allograft survival (Levy, A. E. andAlexander, J. W. (1995) Transplantation 60:405-406), whereasadministration of IL-12 in combination with anti-IL-10 antibodies, whichpromotes Th1 differentiation, enhances skin allograft rejection(Gorczynski, R. M. et al. (1995) Transplantation 60:1337-1341).

Accordingly, the inhibitory methods of the invention can be used tostimulate production of Th2-associated cytokines in transplantrecipients to prolong survival of the graft. The inhibitory methods canbe used both in solid organ transplantation and in bone marrowtransplantation (e.g., to inhibit graft-versus-host disease). Theinhibitory method can involve either direct administration of aninhibitory agent to the transplant recipient or ex vivo treatment ofcells obtained from the subject (e.g., Thp, Th1 cells, B cells,non-lymphoid cells) with an inhibitory agent followed byreadministration of the cells to the subject. The treatment may befurther enhanced by administering other Th2-promoting agents, such asIL-4 itself or antibodies to Th1-associated cytokines, to the recipientin amounts sufficient to further inhibit a Th2-type response.

In addition to the foregoing disease situations, the modulatory methodsof the invention also are useful for other purposes. For example, thestimulatory methods of the invention (i.e., methods using a stimulatoryagent) can be used to stimulate production of Th1-promoting cytokines(e.g., interferon-γ) in vitro for commercial production of thesecytokines (e.g., cells can be contacted with the stimulatory agent invitro to stimulate interferon-γ production and the interferon-γ can berecovered from the culture supernatant, further purified if necessary,and packaged for commercial use).

Furthermore, the modulatory methods of the invention can be applied tovaccinations to promote either a Th1 or a Th2 response to an antigen ofinterest in a subject. That is, the agents of the invention can serve asadjuvants to direct an immune response to a vaccine either to a Th1response or a Th2 response. For example, to promote an antibody responseto an antigen of interest (i.e., for vaccination purposes), the antigenand an inhibitory agent of the invention can be coadministered to asubject to promote a Th2 response to the antigen in the subject, sinceTh2 responses provide efficient B cell help and promote IgG1 production.Alternatively, to promote a cellular immune response to an antigen ofinterest, the antigen and a stimulatory agent of the invention can becoadministered to a subject to promote a Th1 response to the antigen ina subject, since Th1 responses favor the development of cell-mediatedimmune responses (e.g., delayed hypersensitivity responses). The antigenof interest and the modulatory agent can be formulated together into asingle pharmaceutical composition or in separate compositions. In apreferred embodiment, the antigen of interest and the modulatory agentare administered simultaneously to the subject. Alternatively, incertain situations it may be desirable to administer the antigen firstand then the modulatory agent or vice versa (for example, in the case ofan antigen that naturally evokes a Th1 response, it may be beneficial tofirst administer the antigen alone to stimulate a Th1 response and thenadminister an inhibitory agent, alone or together with a boost ofantigen, to shift the immune response to a Th2 response).

This invention is further illustrated by the following example, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference. Additionally, allnucleotide and amino acid sequences deposited in public databasesreferred to herein are also hereby incorporated by reference.

A nucleic acid molecule comprising a mouse T-bet cDNA cloned into theEcoRI site of the pJG4-5 vector was deposited with the American TypeCulture Collection (Manassas, Va.) on Nov. 9, 1999 and assigned DepositNumber PTA-930. A nucleic acid molecule comprising a human T-bet cDNA(prepared from RNA from the human Th1 clone ROT-10) cloned into the PCR2.1-TOPO vector was deposited with the American Type Culture Collection(Manassas, Va.) on Jan. 28, 2000 and assigned Deposit Number PTA-1339.Both deposits were made under the provisions of the Budapest Treaty.

V. Kits of the Invention

Another aspect of the invention pertains to kits for carrying out thescreening assays, modulatory methods or diagnostic assays of theinvention. For example, a kit for carrying out a screening assay of theinvention can include a T-bet-containing indicator composition, meansfor measuring a readout (e.g., polypeptide secretion) and instructionsfor using the kit to identify modulators of biological effects of T-bet.In another embodiment, a kit for carrying out a screening assay of theinvention comprises T-bet deficient cells, means for measuring thereadout and instructions for using the kit to identify modulators of abiological effect of T-bet.

In another embodiment, the invention provides a kit for carrying out amodulatory method of the invention. The kit can include, for example, amodulatory agent of the invention (e.g., T-bet inhibitory or stimulatoryagent) in a suitable carrier and packaged in a suitable container withinstructions for use of the modulator to modulate a biological effect ofT-bet.

Another aspect of the invention pertains to a kit for diagnosing adisorder associated with a biological activity of T-bet in a subject.The kit can include a reagent for determining expression of T-bet (e.g.,a nucleic acid probe for detecting T-bet mRNA or an antibody fordetection of T-bet polypeptide), a control to which the results of thesubject are compared, and instructions for using the kit for diagnosticpurposes.

VI. Immunomodulatory Compositions

Agents that modulate T-bet expression, processing, post-translationalmodifications, or activity are also appropriate for use inimmunomodulatory compositions. Stimulatory or inhibitory agents of theinvention can be used to up or down regulate the immune response in asubject. In preferred embodiments, the humoral immune response isregulated.

T-bet modulating agents can be given alone, or in combination with anantigen to which an enhanced immune response or a reduced immuneresponse is desired.

In another embodiment, agents which are known adjuvants can beadministered with the subject modulating agents. At this time, the onlyadjuvant widely used in humans has been alum (aluminum phosphate oraluminum hydroxide). Saponin and its purified component Quil A, Freund'scomplete adjuvant and other adjuvants used in research and veterinaryapplications have potential use in human vaccines. However, newchemically defined preparations such as muramyl dipeptide,monophosphoryl lipid A, phospholipid conjugates such as those describedby Goodman-Snitkoff et al. J. Immunol. 147:410-415 (1991) resorcinols,non-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether, enzyme inhibitors include pancreatictrypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol canalso be used. In embodiments in which antigen is administered, theantigen can e.g., be encapsulated within a proteoliposome as describedby Miller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated byreference herein, or in lipid vesicles, such as Novasome TM lipidvesicles (Micro Vescular Systems, Inc., Nashua, N.H.), to furtherenhance immune responses.

In one embodiment, a nucleic acid molecule encoding a T-bet molecule orportion thereof is administered as a DNA vaccine. This can be done usinga plasmid DNA construct which is similar to those used for delivery ofreporter or therapeutic genes. Such a construct preferably comprises abacterial origin of replication that allows amplification of largequantities of the plasmid DNA; a prokaryotic selectable marker gene; anucleic acid sequence encoding a T-bet polypeptide or portion thereof;eukaryotic transcription regulatory elements to direct gene expressionin the host cell; and a polyadenylation sequence to ensure appropriatetermination of the expressed mRNA (Davis. 1997. Curr. Opin. Biotechnol.8:635). Vectors used for DNA immunization may optionally comprise asignal sequence (Michel et al. 1995. Proc. Natl. Acad. Sci USA. 92:5307;Donnelly et al. 1996. J. Infect Dis. 173:314). DNA vaccines can beadministered by a variety of means, for example, by injection (e.g.,intramuscular, intradermal, or the biolistic injection of DNA-coatedgold particles into the epidermis with a gene gun that uses a particleaccelerator or a compressed gas to inject the particles into the skin(Haynes et al. 1996. J. Biotechnol. 44:37)). Alternatively, DNA vaccinescan be administered by non-invasive means. For example, pure orlipid-formulated DNA can be delivered to the respiratory system ortargeted elsewhere, e.g., Peyers patches by oral delivery of DNA(Schubbert. 1997. Proc. Natl. Acad. Sci. USA 94:961). Attenuatedmicroorganisms can be used for delivery to mucosal surfaces. (Sizemoreet al. 1995. Science. 270:29)

In one embodiment, plasmids for DNA vaccination can express T-bet aswell as the antigen against which the immune response is desired or canencode modulators of immune responses such as lymphokine genes orcostimulatory molecules (Iwasaki et al. 1997. J. Immunol. 158:4591).

Other means of expressing T-bet, e.g., as described elsewhere herein orknown in the art can also be used. For example, in another embodiment,retroviral vectors are also appropriate for expression of T-betimmunomodulatory compositions. Recombinant retroviral vectors allow forintegration of a transgene into a host cell genome. To transducedividing cells, lentiviral vectors can be used as immunomodulatorycompositions, and are intended to be encompassed by the presentinvention. Lentiviruses are complex retroviruses which, based on theirhigher level of complexity, can integrate into the genome ofnonproliferating cells and modulate their life cycles, as in the courseof latent infection. These viruses include HIV-1, HIV-2, SIV, FIV andEIV. Like other retroviruses, lentiviruses possess gag, pol and envgenes which are flanked by two long terminal repeat (LTR) sequences.Each of these genes encodes multiple polypeptides, initially expressedas one precursor polyprotein. The gag gene encodes the internalstructural (matrix capsid and nucleocapsid) polypeptides. The pol geneencodes the RNA-directed DNA polymerase (reverse transcriptase,integrase and protease). The env gene encodes viral envelopeglycoproteins and additionally contains a cis-acting element (RRE)responsible for nuclear export of viral RNA. The 5′ and 3′ LTRs serve topromote transcription and polyadenylation of the virion RNAs andcontains all other cis-acting sequences necessary for viral replication.Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virions) are missing from the viral genome, the resultis a cis defect which prevents encapsidation of genomic RNA. However,the resulting mutant is still capable of directing the synthesis of allvirion proteins. A comprehensive review of lentiviruses, such as HIV, isprovided, for example, in Field's Virology (Raven Publishers), eds. B.N. Fields et al., 1996.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference.

EXAMPLES

The following experimental procedures were used in the examples:

Mice, Cell Lines, Cytokines, Antibodies and Plasmids

BALB/c mice were obtained from Jackson Laboratories, DO11.10TcR-transgenic mice (Jacobson, N. G., et al. 1995. J. Exp. Med. 181,1755-1762), and MBP TcR-transgenic mice (Lafaille, J. J., 1994. Cell 78,399-408.) have been described. Mice were used at 5-6 weeks of age. Inaddition, C57BL/6 (B6) and BALB/c mice (4-8 weeks old) were purchasedfrom Taconic (Germantown, N.Y.). The generation and screening of T-betdeficient mice have been described (44, 46); and mice used werebackcrossed at least six generations onto the B6 and BALB/c backgrounds.

Cell lines and primary cells were maintained in complete mediumcontaining RPMI 1640 supplemented with 10% fetal calf serum (HyCloneLaboratories), glutamine (2 mM), penicillin (50 units/ml), streptomycin(50 g/ml), Hepes (100 mM) and β-ME (50 μM). Jurkat is a human Th1lymphoma, EL4 a mouse ThO thymoma, NK3.3 a human NK cell line (Ye, J.,1995. J. Leuko. Biol. 58, 225-233; Kornbluth, J., 1982. J. Immunol. 129,2831-2837), YT a human NK cell line (Yodoi, J., 1985. J. of Immuno. 134,1623-1630), AE7 a mouse Th1 clone, D10 a mouse Th2 clone and M12 is a Bcell lymphoma line. Recombinant IL-4 was obtained from DNAX, human rIL-2was obtained from Chiron Corp., rIL-12 was obtained from HoffmanLaRoche, and rIL-18 was purchased from Peprotech, Inc. Monoclonalanti-IL-12, monoclonal anti-IFN-γ and monoclonal anti-IL-4 (11B11) werealso used (Ohara, J. and Paul, W. E. 1985. Nature 315, 333-336). Boththe T-bet polyclonal antisera, produced in rabbits, and the mAb wereraised against full-length recombinant bacterially produced T-bet. ThemAb was produced by fusion of spleen cells from mice to the SP2/O-Ag14myeloma and is of the IgG1 subtype. Expression plasmids included c-Maf(pMex-maf)(Ho, I-C., et al. 1996. Cell 85, 973-983.). NFATp (Hodge, M.R., et al. 1996. Immunity 4, 1-20) and p65, the latter two cloned intothe pCDNA vector.

CD4+ T Cell Purification and In vitro Cultures

CD4+ T cells were purified from lymph nodes (LN) by flow cytometry usingPE-conjugated anti CD4 (RM4-4) (Pharmingen) and sorted using FACS (MoFlo, Becton Dickenson) to 98-99% purity. For in vitro activation2×10⁶/ml CD4+ cells were resuspended in complete medium and activatedwith plate-bound 1 μg/ml anti CD3 (2C11) and 2 g/ml anti CD28(Pharmingen) for 3 days in the presence of 100 units/ml IL2. Cells werethen split 1:4 in complete medium and cultured for 4 days in thepresence of 100 units/ml IL2. On day 7 after primary stimulation, cellswere harvested, washed twice and restimulated at 1×10⁶ cells/ml with 1μg/ml plate-bound anti CD3 for 1, 3 and 6 hours. For Th1 and Th2differentiation cultures, non-transgenic or DO11.10 LN and spleen cellswere pooled, resuspended in 1×10⁶ cells/ml complete medium and culturedunder Th1 (10 mg/ml anti IL4 [11B11], 10 ng/ml rIL12) or Th2 (10 mg/mlanti IFN-γ, 10 ng/ml IL4) conditions with 1 ug/ml plate-bound anti CD3.Cells were split 1:4 on day 3 with complete medium+100 u/ml IL2. On day7, cells were restimulated with 1 g/ml anti CD3 for 4 hours andharvested for RNA preparation (Jacobson, N. G., et al. 1995). J. Exp.Med. 181, 1755-1762). Supernatants were taken at 24 hours to test forcytokines.

Purification and Isolation of Dendritic Cells and Macrophages

Recombinant mouse granulocyte/macrophage-colony stimulating factor(GM-CSF) used to generate bone marrow-derived DCs (bmDC) was produced asculture supernatant from a mouse macrophage cell line (J558L)transfected with the mouse GM-CSF gene (gift of Dr. I. Mellman). L-929cells (gift of Dr. M. Starnbach) were used as the source of L-cell mediato produce BM-derived macrophages

BmDCs were derived by a modification (47) that produces greater numbersof DCs. Briefly, BM cells were cultured in GM-CSF containing DMEM-10media. At day 8, floating cells were collected and purified with CD11c⁺magnetic beads (Miltenyi Biotec, Auburn Calif.) FACS analysis with Absto FITC-I-A^(b) and PE-CD11c⁺ revealed >95% purity. Similarly,macrophages were derived from BM cells cultured in L-cell media (48). Atday 8, adhesive cells were gently scraped off and purified with CD11b⁺magnetic beads. FACS analysis with Abs to FITC-F4/80 and PE-CD14⁺(Pharmigen, San Diego Calif.) revealed >95% macrophages.

Splenic DCs (spDC) and macrophages were isolated by collagenasetreatment (49), and enriched by centrifugation in an Accudenz cellseparation media (Accurate Chemical & Scientific Corp., San Diego,Calif.). T cells and NK cells were subsequently depleted using CD90 andNK1.1 magnetic beads, and DCs were then positively selected with CD11c⁺magnetic beads. In some experiments, DCs were first positively selectedwith CD11c⁺ magnetic beads from collagenase treated spleen, and FACSsorted into subpopulations by staining with FITC-MHC II (I-E/I-A),PE-CD11c⁺, CyC-CD8⁺ or Cyc-CD4⁺ (Pharmingen, San Diego, Calif.).Macrophages were FACS sorted with Abs to PE-CD11b⁺ and FITC-F4/80, orFITC-CD11b⁺ and PE-CD14⁺. To generate activated peritoneal macrophages,1 ml of 3% thioglycollagate (Difco, Irvine, Calif.) was injectedintraperitoneal (i.p.) into young or aged mice; 3 to 5 days later,peritoneal exudates cells were collected, washed in PBS, and cultured inDMEM-10.

DC and Macrophage Stimulation

DCs and macrophages were cultured in DMEM-10 at a concentration of1×10⁶/ml. Cells were stimulated with 10 ng/ml of recombinant mouse IL-12or IL-18 (R&D systems, Minneapolis, Minn.); 1-100 U/ml of rIFN-α/β (NIH,Bethesda, Md.); 20 ng/ml of IL-15 or IL-1 (Peprotech, Rocky Hill, N.J.);1-100 ng/ml of IFN-γ (Peprotech, Rocky Hill, N.J.); 10 ng/ml of IL-21(R&D systems); and LPS (Sigma-Aldrich, St. Louis, Mo.) at 100 ng/ml.

Northern and Western Blot Analysis

Total RNA was isolated from resting and stimulated cells using TRIZOLreagent (Gibco/BRL) and 10 μg of each sample separated on 1.2% agarose6% formaldehyde gels, transferred onto Genescreen membrane (NEN) in20×SSC overnight and covalently bound using a UV Stratalinker(Stratagene). Hybridization of blots was carried out at 42° C. asdescribed (Hodge, M. R., et al. 1996. Immunity 4, 1-20) using thefollowing cDNA probes labeled with 32P: T-bet, γ-actin. Nuclear andcytoplasmic extracts for western blot analysis were prepared from AE7,D10 and NK3.3 cells. Nuclei were isolated as described (Dolmetsch, R.E., et al. 1997. Nature 386, 855-858). Extracted proteins were separatedby 8% PAGE followed by electrotransfer to nitrocellulose membranes andprobed with a mAb specific for T-bet followed by horseradishperoxidase-conjugated goat anti-mouse IgG and enhanced chemiluminescenceaccording to the instructions of the manufacturer (Amersham).

Transient Transfection Assays

EL4 and Jurkat cells were transfected using a Bio Rad electroporator(280V, 975 μF) using 5×10⁶ cells in 0.4 ml RPMI per transfection with 5μg reporter plasmid and 5-10 μg expression plasmid. Luciferase assayswere performed after 24 hrs with the luciferase activity in 20% of eachsample measured as per instructions (Promega). The IFN-γreporter-luciferase construct is derived from the plasmid pB9 whichcontains the entire human IFN-γ gene (P. Gray and D. V. Goeddel. 1982.Nature. 298:859). The pGL2 luciferase gene was inserted into the firstexon of pB9. IL-2-promoter-reporter construct The IL-4 promoter reporterconstruct, IL-4Luc, contains 807 bp upstream of the murine IL-4 gene.

Retroviral Constructs and Transduction

The GFP-RV bicistronic vector has been described (Ouyang, W., et al.1998. Immunity 9:745-755) as has the Phoenix-Eco packaging cell line(Kinoshita, S., et al. 1998. Cell 95, 595-604). The GFP-RV vector wasconstructed by inserting the encephalomyocarditis virus internalribosomal entry sequence (IRES) and the GFP allele into the MSCV2.2retroviral vector (Ouyang, W., et al. 1998. Immunity 9:745-755) orIL-2-MSCV vector. Both vectors express two cDNAs, T-bet and the cDNAencoding GFP, simultaneously using an IRES to initiate translation ofeach mRNA separately. Transfection of the packaging cell line andretroviral transductions of primary T cells were performed essentiallyas described (Ouyang, W., et al. 1998. Immunity 9:745-755).

Intracellular Cytokine Staining and FACS Analysis

Intracellular staining for cytokines was performed as described (Ouyang,W., et al. 1998. Immunity 9:745-755). Primary transgenic ornon-transgenic T cells that had been infected with retrovirus forvarious time periods as indicated were restimulated with PMA (50 ng/mland ionomycin (1 uM) for 2 hours and 10 ug/ml Brefeldin A added for anadditional 2 hours.

Disease Characterization

Hematoxylin and cosin staining of formalin-fixed tissue sections,immunofluorescent studies on OCT-embedded frozen sections, flowcytometry of lymphoid cells, and assays for serum autoantibodies wereperformed as described⁷. Specific antibodies used in this study includedR4-6A2 and XMG1.2 (anti-mouse IFN-γ), BVD4-1D11 and BVD6-24G2(anti-mouse IL-4), MP5-20F3 and MP5-32C11 (anti-mouse IL-6), JES5-2A5and SXC-1 (anti-mouse IL-10), MP1-22E9 and MP1-31G6 (anti-mouse GM-CSF),TN3-19.12 (anti-mouse TNF- ), rabbit anti-TNF-α, HM40-3 (anti-mouseCD40), 1D3 (anti-mouse CD19), and PE-R3-34 (rat IgG1, κ) (BD Pharmingen,San Diego, Calif.); PE-H106.771 (rat IgG1, κ anti-mouse IgG2a) (SouthernBiotechnology Associates, Inc., Birmingham, Ala.); FITC-goat F(ab′)₂anti-mouse IgG (Sigma, St. Louis, Mo.). Anti-DNA activity was determinedby ELISA using high molecular weight mouse DNA (Sigma), and confirmed byimmunofluorescence on Crithidia lucilliae kinetoplasts (AntibodiesIncorporated, Davis, Calif.).

T Cell Assays

Naive CD4+ T cells were purified from spleen and lymph nodes by negativeselection (R&D Systems, Minneapolis, Minn.) and stimulated for 48-72hours in RPMI/10% with 1 μg/mL anti-murine CD28 (37.51) antibody and 1μg/mL plate-bound anti-murine CD3 (145-2C11) antibody (BD Pharmingen).Cytokine production was evaluated in culture supernatants by ELISA (BDPharmingen, San Diego, Calif.). Proliferation was measured by BrdUincorporation (Amersham Pharmacia Biotech, Piscataway, N.J.). Apoptosiswas evaluated by exposing the cells for 24 hours to 20 μg/mL solubleanti-mouse CD3 and anti-mouse CD28, 5 μg/mL dexamethasone (Sigma), or1200 J UV irradiation in a Stratalinker (Stratagene, La Jolla, Calif.),followed by evaluation by the CaspACE™ Assay System (PromegaCorporation, Madison, Wis.).

Immunoglobulin Assays

For in vitro analyses, purified mature B cells were isolated from spleenand lymph nodes by magnetic CD43 depletion (Miltenyi Biotec, Auburn,Calif.) and stimulated in RPMI/10% with 25 μg/mL LPS (Sigma)supplemented with recombinant murine IL-4 at 10 ng/mL, IFN-γ at 100ng/mL, human TGF-β1 at 1 ng/mL (PeproTech, Rocky Hill, N.J.), or murineIFN-γ at 100 U/mL (R&D Systems, Minneapolis, Minn.). For retroviralinfection studies, purified CD43-depleted mature B cells were stimulatedby 25 μg/mL LPS for 24 hours, followed by infection by a T-bet-GFP orcontrol-GFP retrovirus². Quantification of serum immunoglobulin isotypesin serum or culture supernatants was performed as previously described.Germline and post-switch transcripts were determined by RT-PCR asdescribed previously.

Real-Time PCR, ELISA, and Western Blot Analysis

RNA was isolated with Trizol (Sigma-Aldrich) from un-stimulated andstimulated DCs and macrophages. cDNA synthesis was performed with 1 ugof total RNA using oligo (dT)15 primer (SEQ ID NO: 17), 20 nM of eachdNTP, 0.1 M DTT, 1× first-stranded buffer, SuperScript II, and RNaseOUT(all from Invitrogen, Carlsbad, Calif.). Semiquantitative RT-PCR todetermine the levels for T-bet, IFN-γ, TNF-α, IL-12 subunits p40 andp35, and other inflammatory cytokines, was performed as described byOverbergh et al (50). TaqMan universal PCR master mix was used for allreactions (AB Applied Biosystems, Branchburg, N.J.). Sequences ofprimers and TaqMan probe for most cytokines including β-actin are asdescribed (51). Expression levels for the gene of interest are reportedrelative to β-actin abundance. Protein levels of IFN-γ and IL-12p40 weredetected by ELISA from harvested supernantants of stimulated DCs andmacrophages (Pharmigen). To detect the expression of T-bet protein,whole extracts were collected from DCs stimulated for different timeperiods and detected by immunoblot analysis as previously described(45).

Example 1 Cloning of a Novel Transcription Factor, T-Bet

Since the Th1-specific region of the IL-2 promoter had been welllocalized (Brombacher, F., et al. 1994. Int. Immunol. 6:189-197; Rooney,J., et al. 1995. Mol. Cell. Biol. 15, 6299-6310; Lederer, J. A., et al.1994. J. Immunol. 152, 77-86; Durand, D., et al. 1988. Mol. Cell. Biol.8, 1715-1724; Hoyos, B., et al. 1989. Science 244, 457-450), a yeast onehybrid approach using an IL-2 promoter-reporter and a cDNA library madefrom the OF6 Th1 clone was chosen to identify Th1 specific transcriptionfactors. To validate this approach, the Th2-specific region of the IL-4promoter was expressed in yeast and demonstrated to be transactivated bythe introduction of c-Maf, but not by several other transcriptionfactors (eg NFAT). C-Maf transactivation did not occur when the c-Mafresponse element (MARE) was mutated. Thus, the yeast one hybrid approachwas utilized.

The EGY48 yeast strain was stably integrated with the IL-2promoter/histidine construct and transformed with a cDNA library madefrom an anti-CD3 activated Th1 cell clone, OF6. Of 5.6×10⁶ clonesscreened, 488 were positive in primary screening. Of the 210 clonestested during the secondary screen, 72 proved to be specific for theIL-2 promoter. To reduce the number of positive clones, we hybridizedthe yeast clone cDNA with cDNAs that were differentially expressed inTh1 and Th2 cell lines. These Th1-Th2 and Th2-Th1 cDNAs were made usingthe Clontech PCR select kit, radiolabeled and initially used in a pilotexperiment to screen the 16 most strongly positive yeast clones. Ofthose 16 clones, 8 were positive with the Th1 (PL17) specific cDNAproduct probe and not with the Th2 (D10) specific cDNA product probe.Representational difference analysis (RDA; e.g., Lisitsyn. 1993.Science. 259:946; O'Neill and Sinclair. 1997. Nucleic Acids Res.25:2681; Hubank and Schatz. 1994. Nucleic Acids Research. 22:5640;Welford et al. 1998. Nucleic Acids Research. 26:3059) with Th1-Th2 probeon 16 positive clones with control hybridization of the probe to IL-2,IFN-γ and IL-4 was performed. The specificity of the Th1 and Th2subtracted cDNA probes is demonstrated by their detection of IL-2 andIFN-γ versus IL-4 respectively.

Restriction enzyme analyses and sequencing data revealed that all 8 ofthe clones were related. They fell into three groupings based ondifferences in the 5′ and 3′ untranslated regions, each of thesecategories representing an independent cDNA molecule. Comparing thesequence of these clones with the NCBI GenBank Sequence Database yieldedhomology with the T-box family of transcription factors. FIG. 1 shownthe nucleotide and amino acid sequences of T-bet.

Example 2 T-Bet Shares a Region of Homology with the T-box FamilyMembers T-Brain and Eomesodermin

Brachyury or T is the founding member of a family of transcriptionfactors that share a 200 amino acid DNA-binding domain called the T-box(reviewed in (Smith, J. 1997. Current Opinion in Genetics & Development7, 474-480; Papaioannou, and Silver. 1998. Bioessay. 20:9; Meisler, M.H. 1997. Mammalian Genome 8, 799-800.). The Brachyury (Greek for ‘shorttail’) mutation was first described in 1927 in heterozygous mutantanimals who had a short, slightly kinked tail (Herrmann, B. G., 1990.Nature 343, 617-622). There are now eight T-box genes in the mouse notincluding Brachyury. These include Tbx1-6, T-brain-1 (Tbr-1) and now,T-bet, each with a distinct and usually complex expression pattern. TheT-box family of transcription factors is defined by homology of familymembers in the DNA binding domain. The T-bet DNA binding domain(residues 138-327 of murine T-bet) is most similar to the T-box domainsof murine T-brain and Xenopus eomesodermin and thus places T-bet in theTbr1 subfamily of the T-box gene family. The human homologue of themurine T-bet protein is approximately 88% identical to the mouse T-bet.FIG. 1A was derived using a Lipman-Pearson protein alignment (with Gpenalty set at 4 and gap length penalty set at 12. The similarity indexwas calculated to be 86.6; the gap number 2, the gap length 5, and theconsensus length 535). T-bet shares a region of homology with the T-boxfamily members T-brain and eomesodermin. The murine T-bet DNA bindingdomain is most similar to the T-box domains of murine T-brain andXenopus eomesodermin. There is approximately 69% amino acid identitybetween the three T-box regions. T-bet bears no sequence homology toother T-box family members outside of the T-box domain.

Example 3 T-Bet Binds to and Transactivates Consensus T-Box Sites andhas Functionally Important Domains that Map to Both 5′ and 3′ Regions

Recombinant T-bet protein binds to consensus T-box sites and to theT-bet site in the IL-2 promoter, and a complex present in nuclearextracts from anti-CD3-stimulated AE7 Th1 cells binds specifically to aconsensus (GGGAATTTCACACCTAGGTGTGAAATTCCC) (SEQ ID NO: 5) T-boxoligonucleotide probe. To test for activity of T-bet in T cells, thefollowing experiments were performed. Jurkat Th1 cells werecotransfected with T-bet and a luciferase reporter construct. FIG. 2Ashows the basal level (open bars) and the PMA (50 ng/ml) plus ionomycin(1 uM) induced (closed bars) promoter activity in Jurkat cells of aluciferase reporter construct containing a minimal thymidine kinase (TK)promoter with or without 4 copies of the consensus T-box site. Eachreporter construct was co-transfected with empty pCDNA vector or pCDNAcontaining the full-length T-bet cDNA as indicated in the figure. Thedata shown are representative of three independent experiments. FIG. 2Bshows Jurkat cells transiently transfected with the luciferase reporterconstruct containing the minimal TK promoter and multimerized consensusT-box sites and pCDNA vector containing the indicated regions of theT-bet cDNA diagrammed at the left of the bar graph. Luciferase activitywas measured 24 hours post-transfection. The experiment was repeatedthree times with similar results. The basal level (open bars) and thePMA (50 ng/ml) plus ionomycin (1 uM) induced (closed bars) promoteractivity obtained demonstrate that T-bet is active in T cells, and thatits activity can be further increased upon stimulation.

Example 4 T-Bet Expression in T Cells is Restricted to the Th1 Subsetand Regulated by Signals Transmitted via the TcR

T-bet was isolated from a Th1 cDNA library and a multiple organ Northernblot analysis revealed T-bet transcripts only in lung, thymus and inperipheral lymphoid organs.

FIG. 3A shows that T-bet is preferentially expressed in double negative(DN) thymocytes, not in double positive (DP) or single positive (SP)cells. Northern blot analysis of total cellular RNA isolated from Th1cell clones (AE7 and D1.1) or Th2 clones (D10 and CDC35) that weretreated with media or with plate-bound anti-CD3 (2C11) for 6 hoursrevealed T-bet transcripts only in the Th1 clones. Total cellular RNAwas isolated from Th1 cell clones (AE7 and D1.1) or Th2 clones (D10 andCDC35) that were treated with media or with plate-bound anti-CD3 (2C11)for 6 hours. Total RNA was also isolated from M12 (B-cell lymphoma andEL4 (T-cell thymoma) treated with media or with PMA (50 ng/ml) andionomycin (1 uM) for 6 hours. Northern blot analysis was performed with10 ug of total RNA per lane using standard procedures and probed usingthe full-length T-bet cDNA. T-bet is preferentially expressed in Th1clones. Further, the level of T-bet expression was augmented by signalstransmitted via the TcR as evidenced by the induction of T-bettranscripts by anti-CD3. T-bet transcripts were not detected in M12, aB-cell lymphoma, in the Th1 lymphoma Jurkat or in EL4, a Th0-cellthymoma either when these cells were treated with media or with PMA (50ng/ml) and ionomycin (1 uM) for 6 hours.

To determine protein levels of T-bet in primary T cells, DO11.10 TcRtransgenic splenocytes were cultured under Th1 or Th2 polarizingconditions. At 72 hours the cells were expanded 3-fold in fresh mediumwith 200 U/ml IL-2. On day 7 after primary stimulation, nuclear andcytosolic extracts were prepared from resting or PMA/ionomycin activated(1 hr) bulk culture DO11.10 Th1 and Th2 cells. Nuclear extracts werealso prepared from resting M12, EL4, Jurkat, NK3.3, and YT cells. Asshown in FIG. 3C, among the cell lines, T-bet protein was present in YTcells only. FIG. 3C shows T-bet protein is restricted to Th1 cells andNK cells. Western blot analysis was performed on nuclear and cytosolicextracts prepared from resting or PMA/ionomycin activated (1 hr) bulkculture DO11.10 Th1 and Th2 cells as above. Briefly, D011.10 Tcrtransgenic splenocytes were activated with OVA peptide (323-339) at3×106 cells/ml in the presence of 10 ng/ml IL-12 and 10 ug/ml anti-IL-4(11B11) to promote Th1 phenotype development, or 10 ng/ml IL-4 and 10ug/ml anti-IFN-γ to promote Th2 phenotype development. At 72 hours thecells were expanded 3-fold in fresh medium with 200 U/ml IL-2. On day 7after primary stimulation, nuclear and cytosolic extracts were preparedfrom resting or PMA/ionomycin activated (1 hr) bulk culture D011.10 Th1and Th2 cells. Nuclear extracts were also prepared from resting M12cells, EL4, Jurkat, NK3.3, and YT. 30 ug of nuclear and cytosolicextracts were separated by SDS-PAGE (8% gel), transferred tonitrocellulose, and probed with an anti T-bet antisera. In primary Tcells, T-bet protein is selectively expressed in T cells driven along aTh1 but not a Th2 pathway, consistent with the Northern blot analysis ofT cell clones and primary T cells shown above.

A monoclonal antibody (mAb) specific for T-bet allowed the directvisualization of T-bet protein by FACS analysis. FIG. 3D shows thatT-bet can be visualized by FACS in activated AE7 Th1 cells. D10 (Th2) orAE7 (Th1) cells were treated with media or PMA (50 ng/ml) plus ionomycin(1 uM) for 2 hours and 2 uM monensin for an additional 3 hours. Cellswere washed with PBS, fixed in 4% paraformaldehyde, permeabilized with0.5% saponin, and stained with media (dashed line) or an IgG1 isotypecontrol antibody (dotted line) or an affinity-purified anti-T-betmonoclonal antibody 3D10 (solid line) followed by goat anti-mouseIgG1-PE staining. Cells were analyzed by flow cytometry on aFACSCalibur. Mouse monoclonal antibodies were raised against full lengthbacterially produced T-bet. T-bet protein was not detectable in D10cells, was present at low levels in unstimulated AE7 cells and waspresent at increased levels in stimulated AE7. Taken together, theexperiments detailed here demonstrate that in T cells, T-bet isselectively expressed in Th1 cells where its level of expression isregulated by signals stemming from the TcR.

Example 5 T-Bet Expression Correlates with IFN-γ Induction in NK and BCells

The Th1-limited expression of T-bet coupled with its isolation by virtueof binding to a T-box site in the IL-2 promoter suggested that T-betmight activate the transcription of the IL-2 gene. However, it waspuzzling that two IL-2-producing cell lines, Jurkat and EL4, did notexpress T-bet, while the NK cell line YT, which produces IFN-γ but notIL-2, did express T-bet. Further, preliminary experiments did notdemonstrate transactivation of the IL-2 gene by T-bet, despite thepresence of an excellent T-box site in the IL-2 promoter. OtherTh1-specific cytokines include IFN-γ, TNFα and LT. The expression ofT-bet correlated well with the expression of IFN-γ. Further, a T-boxsite was found to be present in the third intron of the human IFN-γgene. This was especially noteworthy since a Th1-specific DNaseIhypersensitivity site had recently been mapped to this region.

To examine the possibility that T-bet controlled the expression of theIFN-γ gene, the expression of T-bet and the expression of IFN-γ in cellsother than Th1 cells was measured. IFN-γ is expressed in natural killer(NK) cells at low levels and is induced to high levels upon treatmentwith IL-2 and IL-12 (Kornbluth, J., et al. 1982. J. Immunol. 129:2831;Ye et al. 1995. J. Leuko. Biol. 58:225). Therefore, the NK3.3 cell linewas treated for 24 h with IL-2, IL-12 and IL-2 plus IL-12, lysatesprepared and western blot analysis performed with T-bet mAb as above.FIG. 4 b demonstrates coordinate induction of T-bet protein andsecretion of IFN-γ in NK3.3 cells. The NK3.3 cell line was treated for24 h with reagents, IL-2, IL-12 and IL-2 plus IL-12, known to induceIFN-γ in NK cells, lysates prepared and western blot analysis performedwith T-bet mAb as above. ELISA was performed on supernatants harvestedfrom the cells.

B cells, which do not produce IFN-γ at baseline, can be driven toproduce large amounts of IFN-γ upon treatment with anti-CD40 antibodyand a combination of IL-12 and IL-18 (Yoshimoto, T., 1997. Proc. Natl.Acad. Sci. USA 94, 3948-3953). Purified B cells were treated for 72 hwith anti-CD40 mAb, rIL-12 and rIL-18, RNA isolated and Northern blotperformed using the T-bet cDNA as above. FIG. 4A shows induction ofT-bet mRNA in B cells treated with this combination of reagents, and theinduction of IFN-γ transcripts in these cells was confirmed. Inconclusion, while neither cell type expresses T-bet constitutively, bothNK3.3 cells and B cells can be induced to do so under conditions whichalso result in IFN-γ production. Thus, the pattern of expression ofT-bet correlates well with the transcription of the IFN-γ gene.

Example 6 T-Bet Transactivates the IFN-γ Gene in Th Cells

Very little is yet known about the regulatory regions of the IFN-γ gene.In particular, the regions of the gene that direct its tissue-specificexpression have not been identified in vitro or in vivo. It has beendemonstrated that reporter constructs containing 500 bp or 3 kb ofupstream sequence are expressed in both Th1 and Th2 cells (Young, H. A.,1994. J. of Immuno. 153, 3603-3610). ATF-2, NF B, AP-1 and Stat4 sitesin the IFN-γ promoter or introns are thought to be functionallyimportant, but clearly are not responsible for tissue-specificexpression (Young, H. A., 1994. J. of Immuno. 153, 3603-3610; Sica, A.,1997. J. Biol. Chem. 272, 30412-30420; Penix, L., 1993. J. Exp. Med.178, 1483-1496; Penix, L. A., 1996. J. Biol. Chem. 271, 31964-31972).Similarly, although Th1-preferential DNaseI hypersensitive sites havebeen noted both in the first and third introns, the relevant ciselements located in these introns have not been identified (Young, H.A., et al. 1994. J. of Immunol. 153, 3603-3610; Agarwal, S. and Rao, A.1998. Immunity 9, 765-775). Therefore, a reporter construct containingthe entire IFN-γ gene was utilized for these studies. The IFN-γ reportergene used includes 3 kb of upstream sequence, the entire coding sequencewith all three introns, and 1.5 kb of downstream (Xu, X., et al 1996.Science 273, 794-796).

The activity of a luciferase reporter construct containing 9 kb of theIFN-γ gene in the Jurkat human Th1 lymphoma and the mouse EL4 Th0tyymoma was tested. Each reporter construct (10 ug) was co-transfectedwith empty pCDNA vector or pCDNA containing the full-length T-bet cDNA,c-Maf, NFATp or p65 (10 ug). The constructs also include the −400 to −40IL-2 and IL-4 promoter luciferase reporters.

The Th0 mouse T cell thymoma EL4, which produces IL-2 and IL-4 but notIFN-γ was transfected with a T-bet cDNA expression plasmid and theIFN-γ-luciferase reporter (FIG. 5). Introduction of the T-bet expressionplasmid resulted in (approximately 20-30 fold) transactivation of theIFN-γ gene compared to empty vector alone. This was in contrast to theabsence of transactivation by two other factors, the Th2-specifictranscription factor c-Maf and the Th non-selective transcription factorNFAT. Interestingly, although the NF B family member, p65, did nottransactivate the IFN-γ reporter on its own, cotransfection of T-bet andp65 resulted in a synergistic activation.

Examination of the IL-2 promoter was also made using a region of thepromoter known to be Th1-specific (Lederer, J. A., et al. 1994. J.Immunol. 152, 77-86). T-bet repressed the activity of the IL-2 promoterapproximately 10 fold. This was especially apparent upon activation ofthe promoter by PMA and ionomycin. As before, substantialtransactivation of the IFN-γ gene was noted. T-bet activity was specificfor the IL-2 and IFN-γ genes since no effect on transactivation of anIL-4 promoter (FIG. 5) or a TNF-α promoter was present. These datademonstrate that T-bet specifically activates the transcription of theIFN-γ gene, and represses the transcription of the IL-2 gene.

To examine endogenous gene expression, EL4 cells were transientlytransfected with T-bet or empty vector, and IFN-γ production measured byELISA 48 hours after stimulation with PMA/ionomycin (FIG. 5). Consistentwith the transactivation data shown above, ectopic expression of T-betin EL4 cells led to measurable IFN-γ production while transfection withvector control did not result in detectable IFN-γ.

Example 7 Retroviral Gene Mediated Transfer of T-Bet into Primary ThCells Results in Increased IFN-γ Production

The experiments described above argue strongly for a critical role ofT-bet in controlling the transcription of the IFN-γ gene.

A bovine collagen-specific Th0 hybrid was transduced with retroviralconstructs containing T-bet GFP or GFP only under the control of the TcRinducible IL-2 promoter. Transduced populations were FACS sorted on GFPtwice, rested and then stimulated with anti-CD3 and supernatantscollected at 60 hours to measure cytokine production by ELISA. (FIG. 6).Control retroviral vectors which had not effect included anti-senseT-bet.

To further test whether T-bet is responsible for the tissue-specificexpression of IFN-γ, retroviral gene mediated transfer of T-bet intoprimary T cells, both non-transgenic and TcR transgenic, was performed.Two different bicistronic retroviruses expressing both T-bet and GFPwere used. The first expresses T-bet under the control of an IL-2inducible promoter, and the second expresses T-bet under control of anMSCV LTR. Similar results were obtained with both constructs.

BALB/c CD4 T cells were infected after 36 hours of primary activation byanti-CD3 plus anti-CD28, harvested on day 7 and intracellular IFN-γ andIL-2 staining performed 5 hours after stimulation with PMA and ionomycinas described in Experimental Procedures. Data are shown as two-colorplots showing GFP expression (FL1) versus intracellular cytokine (FL2)of events gated on expression of CD4. Primary T cells from MBP TcRtransgenic mice were stimulated using MBP (Ac1-11) at 6 uM and infectionperformed on day 1 with IL-2/GFP and IL-2/T-bet/GFP. On day 7, cellswere sorted for GFP expression, rested for 1 day and then intracellularcytokine analysis performed after a 5 hour stimulation with PMA andionomycin.

Naive MBP-transgenic or non-transgenic BALB/c CD4 T cells were activatedwith MBP 1-11 and anti-CD3 under non-polarizing conditions and wereinfected with retrovirus on day 1 after primary activation as described(Ouyang, W., et al. 1998. Immunity 9:745-755). Cells were cultured for 7days and then GFP expression measured to determine percentage of cellsinfected. GFP positive cells were sorted and cytokine productionmeasured by intracellular staining after an additional 4 hoursstimulation with PMA plus ionomycin.

Transduction of both MBP-TcR transgenic and non-transgenic T cells withT-bet resulted both in an impressive increase in the number of cellsproducing IFN-γ and in the amount of IFN-γ produced per cell as comparedto cells transduced with GFP alone. (FIG. 7).

Naive Thp cells, early after stimulation, produce large amounts of IL-2,which is then gradually replaced in polarized Th cells by the effectorcytokines IFN-γ and IL-4. Polarized Th1 cells do continue to produceIL-2 but at amounts considerably less than naïve Thp. Polarized Th2cells shut off the production of IL-2. T-bet transduced Th cellsproduced somewhat less IL-2 than GFP/RV control transduced cells,consistent with the repression of IL-2 promoter transactivation by T-betthat we observed in EL4 cells. The repression of IL-2 by T-bet isconsistent with a function for T-bet in driving lineage commitment froma naive precursor cell into a fully differentiated effector cell.

Example 8 T-Bet Activates IFN-γ and Represses IL-4 Production inDeveloping Th2 Cells

The experiments above demonstrate that T-bet can direct unskewed Thcells into the Th1 pathway. The T-bet could force Th cells to directtheir genetic program along a Th1 pathway even in the presence ofstimuli that would ordinarily drive them into the Th2 pathway wastested. In the experiments in FIG. 8, BALB/c CD4+ T cells were activatedwith anti-CD3 and anti-CD28 in the presence of rIL-4 and antibodies toIFN-γ and IL-12, retroviral infection performed at 36 hours, cellsexpanded with IL-2, GFP positive cells sorted on day 7 and cytokineproduction measured by intracellular staining after an additional 4hours stimulation with PMA plus ionomycin. Transduction with GFP-RValone resulted in a population that contained 13.4% IL-4-producing cellsand 0.9% IFN-γ producers (FIG. 8). As expected, the Thp cells are notyet fully polarized at this time. Introduction of T-bet/GFP/RV produceda substantial shift of Thp into the Th1 pathway as evidenced by thelarge number of cells (50%) producing IFN-γ and the reduced number ofcells producing IL-4 (3.5%), even under conditions (rIL-4 andanti-IL-12) that inhibit Th1 differentiation. Thus, T-bet can overcomethe Th2-promoting signals delivered by cytokines to drive developing Thcells into the Th1 pathway.

Example 9 T-Bet Redirects Polarized Th2 Cells into the Th1 Pathway

It has been demonstrated that reversibility of Th1 and Th2 populationsis lost after long-term stimulation under polarizing conditions.Reversibility is largely abrogated after one week and is completely lostafter 3 weeks (Murphy, E., et al. 1996. J. Exp. Med. 183, 901-913.). Todetermine whether T-bet could redirect the commitment of a purepopulation of already polarized Th2 cells, CD4+ T cells were cultured asabove and retroviral gene transduction performed at day 9 of culture. InTh cells cultured for 9 days under Th2 polarizing conditions, controlGFP/RV-transduced cells are virtually all IL-4 and IL-5 producers (23%and 11%) with barely detectable IFN-γ producer cells (6%) (FIG. 9).Thus, as expected, almost complete polarization had occurred.Remarkably, introduction of T-bet into these fully polarized Th2 cellsredirected or converted them into polarized Th1 cells as evidenced bothby the induction of IFN-γ expression and the loss of IL-4 and IL-5expression. This conversion occurred in the presence of exogenous IL-4.Fully 77% of T-bet-transduced Th2 cells now produced IFN-γ while thepercentage of cells producing IL-4 and IL-5 has been reduced to 13% and1% respectively. These T-bet-transduced cells are therefore not Th0cells that produce both IFN-γ and IL-4. Therefore, T-bet has not simplyinduced IFN-γ expression in Th2 cells but has actually reprogrammed Th2cells into the opposing Th1 subset.

Example 10 T-Bet also Redirects Polarized Tc2 Cells into the Tc1 Pathway

Although most attention has focused on the CD4+ T lymphocyte, it isapparent that cytotoxic CD8+ T cells also may also be divided intoIFN-γ-producing (Tc1) and IL-4-producing (Tc2) subsets. The ability ofT-bet to redirect fully polarized Tc2 cells into a Tc1 pathway wastested. Purified CD8+ T cells were therefore differentiated in cultureunder Tc2 polarizing conditions for 9 days to accomplish fulldifferentiation. FIG. 10 demonstrates that T-bet transduced Tc2 cells,similar to T-bet transduced CD4 Th2 cells have been reprogrammed toproduce IFN-γ (85% versus 15%) and to repress the production of IL-4 andIL-5 (3% versus 34% and 1% versus 45% respectively). Thus, T-bet canconvert fully differentiated CD8+ Tc2 cells to Tc1 cells.

Example 11 T-Bet is Tyrosine Phosphorylated

To determine whether T-bet is a tyrosine phosphorylated protein, wholecell lysates from AE7 Th1 cells were prepared after incubation for 0, 5,10, 30 minutes with pervanadate. Lysates were immunoprecipitated withanti-T-bet antiserum, separated by SDS-PAGE (8% gel), transferred tonitrocellulose, and probed with an anti-phosphotyrosine mAB 4G10.Following exposure, blots were stripped and reprobed with anti-T-betantisera. As shown in FIG. 11, T-bet is clearly a tyrosinephosphorylated protein in T cells.

Example 12 Creation of a Dominant Negative T-Bet Molecule

Chimeric cDNA molecules were made with the T-bet DNA binding domain(residues 138-327) and the repressor domain of the Drosophila proteinengrailed. The engrailed protein is a powerful, active repressor oftranscription (Taylor, D., 1996. Genes Dev. 10, 2732; Li, J., Thurm, H.,et al. 1997. Proc. Natl. Acad. Sci. USA 94, 10885). The T-bet-engrailedconstruct in vitro using a multimerized T-box consensus site/TK minimalpromoter luciferase reporter construct. As shown in FIG. 12,T-bet/engrailed specifically and significantly represses the ability ofwild type T-bet to transactivate a T-box reporter construct at a 5:1ratio, and does not repress transactivation of an NFAT or NFkB reporterby NFATp and p65 expression constructs respectively.

Example 13 T-Bet is Required for Interferon-γ Production and Th1 LineageCommitment

A. Generation of T-Bet Deficient Mice

To definitively address the role of T-bet in IFN-γ production and Th1lineage commitment, T-bet deficient mice were generated. The T-bet genewas disrupted by homologous recombination by replacing the first exon,500 bp of upstream sequence, 1 kb of intronic sequence with the neomycinresistance gene. Germline chimeric animals generated from the targetedTC1 embryonic stem cell clone produced heterozygous mice which were thenintercrossed to obtain mice homozygous for the T-bet mutation(T-bet^(−/−)). T-bet deficient mice were born at the expected Mendelianratios and were phenotypically normal and fertile. To confirm that theT-bet mutation inactivated the T-bet gene, total RNA or total proteinlysates were isolated from resting or PMA/ionomycin activated CD4⁺ Tcells from wild type littermate controls, and heterozygous or homozygousmutant mice. T-bet expression was not detected by Northern or Westernblot analysis in T-bet^(−/−) CD4⁺ T cells and was present at a reducedlevel on T-bet^(+/−) heterozygotes.

Flow cytometric analysis of thymocytes, splenocytes and lymph node cellsfrom littermate controls and T-bet^(−/−) mice revealed no abnormalitiesin expression of CD3, CD4, CD8, B220 nor in the composition oflymphocyte populations within each peripheral lymphoid organ. Thus,T-bet is not required for normal thymocyte maturation or mature T/B cellhoming to peripheral organs.

B. T-bet Controls IFN-γ Production and Th1 Lineage Commitment

The cytokine production profiles from CD4⁺ T cells from T-bet deficientmice were examined. CD4⁺ T cells were purified from the lymph nodes ofT-bet^(−/−), T-bet^(+/−) and T-bet^(+/+) mice to yield a population of95% pure naïve CD4⁺/Mel14⁺ T cells. A striking decrease in IFN-γproduction by T-bet^(−/−) CD4⁺ T cells was observed as measured by ELISA72 hrs after anti-CD3/CD28 stimulation as compared to wild typelittermate control T-bet^(+/+) CD4⁺ T cells. A corresponding increase inIL-4 production was observed in T-bet^(−/−) CD4⁺ T cells. These resultsdemonstrate that T-bet deficient cells produce Th2 type cytokines duringa primary stimulation under neutral conditions (in which no cytokines oranti-cytokine antibodies were added).

To determine if this was an immediate effect on cytokine production oran effect on T helper cell differentiation, CD4⁺ T cells were purifiedfrom the lymph nodes of T-bet^(−/−), T-bet^(+/−) and T-bet^(+/+) miceand stimulated through the TCR under neutral conditions or under Th1 orTh2 inducing conditions to generate effector T helper cells. Uponrestimulation with anti-CD3, cytokine production was measured by ELISA.T-bet^(−/−) CD4⁺ T cells produced dramatically less IFN-γ than thecontrol T-bet^(+/+) CD4⁺ T cells with a concomitant increase in IL-4 andIL-5 production. This effect was seen even when the cells werestimulated in the presence of Th1 inducing conditions. Uponrestimulation, these cells produced very low levels of IFN-γ and couldnot suppress production of IL-4 and IL-5. Thus, even under Th1 inducingconditions, T-bet^(−/−) CD4+ T cells default toward the Th2 lineage.These results were confirmed by ICC, an assay that allows for theexamination of each IFN-γ producing cell, which showed a strikingdecrease in the number of IFN-γ producing cells in the absence of T-bet.We conclude that T-bet controls not only immediate cytokine productionbut also has a profound effect on T helper effector function.

Interestingly, heterozygous T-bet^(+/−) CD4⁺ T cells displayed anintermediate phenotype of cytokine production. It is possible that theabsence of one allele of T-bet, with a corresponding decrease in T-betprotein, resulted in all CD4 T cells producing half as much IFN-γ aswild-type cells. Alternatively, there might be an exquisite sensitivityto threshold levels of T-bet with half as many cells producing wildtypelevels of IFN-γ. To distinguish between these possibilities, ICC assayswere performed. Cells were stimulated under Th1 inducing conditions for7 days, then restimulated with PMA/ionomycin and analyzed forintracellular IFN-γ. This revealed that 85% of wildtype Th1 cells wereIFN-γ producers while a striking decrease was observed in T-betdeficient Th1 cells (9%) and an intermediate phenotype observed in theheterozygous T cells (46%). Therefore the function of T-bet incontrolling IFN-γ production is highly dosage sensitive, a finding thatis consistent with the known function of other T-box family genes inwhich haploid insufficiency of Tbx3 and Tbx5 leads to the geneticdisorders, Ulnar Mammary and Holt-Oram syndromes, respectively. Anotherpossibility is monoallelic, rather than biallelic expression of T-bet asdocumented for certain cytokine genes (e.g. IL-2 and IL-4).

D. Conclusion

The analysis of the immune system in mice that lack T-bet, as describedabove, firmly establishes T-bet as a transcription factor that isrequired for Th1 lineage commitment. Further, it is clear that onemechanism by which this occurs is the control, of IFN-γ genetranscription by T-bet in vivo. Mice that lack T-bet do not develop arobust Th1 compartment as evidenced by the failure of CD4 T cells toproduce the hallmark Th1 cytokine, IFN-γ even upon deliberatepolarization. A large number of transcription factors have beenimplicated in the control of the IFN-γ gene. ATF-2, NFκB, AP-1, YY1,NF-AT and Stat sites in the IFN-γ promoter or introns are functionallyimportant in vitro, but are not responsible for the tissue-specificexpression of IFN-γ (Young et al., 1994; Sica et al., 1997; Penix etal., 1996; Xu et al., 1996; Sweetser et al., 1998), nor do theyselectively control IFN-γ in vivo. Here we have demonstrated that T-betis selectively required for IFN-γ production in CD4+ T cells and NKcells in vivo. Given the pathogenic role of Th1 cells in autoimmunityand cancer, and their protective role in asthma, these observations haveclear implications for the treatment of human disease.

Example 14 T-Bet is Required for Interferon-γ Production and LineageCommitment in CD4 and in Antigen Activated but not Anti-CD3 ActivatedCD8 T Cells

T-bet is expressed in both CD4 and CD8 T cells. To determine whetherT-bet is involved with IFN-γ production in both CD4 and CD8 T cells, thefollowing experiments were performed. Purified CD4 and CD8 T cells werestimulated for 72 hrs with plate bound anti-CD3, anti-CD28, rIL-12 andrIL-18, RNA prepared and northern blot analysis performed using T-bet,IFN-γ, and HPRT probes. CD8 T cells and CD4 T cells purified fromT-bet^(−/−), T-bet^(+/−) and T-bet^(+/+) LN were stimulated withplate-bound anti-CD3 and anti-CD28 for 7 days. ICC analysis wasperformed after 5 hours stimulation with PMA (50 ng/ml) and ionomycin (1uM). IFN-γ production was measured by ELISA 24 hrs after restimulationwith anti-CD3/anti-CD28. CTL precursors from T-bet^(+/+) or ^(−/−)splenocytes were primed in vitro with Concanavalin A (5 ug/ml or platebound anti-CD3/anti-CD28 and 100 U/ml hIL-2 for 5 days (32). On day 5CD8 T cells (H-2^(b)) were purified by positive selection using MACSpurification and incubated for 4 hours with ⁵¹Cr labeled P815 (H-2^(d))allogeneic target cells at the indicated effector to target ratios.

The results of these experiments demonstrated that in contrast to CD4 Tand NK cells, T-bet is not involved in controlling IFN-γ production inthe other major subset of T cells, the cytotoxic CD8 T cell when thiscell is stimulated via the T cell receptor and costimulatory CD28receptor with antibodies to CD3 and CD28. However, as described inExample 28, T-bet does control the production of IFN-γ fromantigen-activated CD8 cells.

Example 15 T-Bet Regulates IgG Class Switching and PathogenicAutoantibody Production

Because of its role in Th1 responses, it is likely that T-bet would playa critical role in systemic autoimmune syndromes like lupus, which relyheavily upon Th1 T cells for pathogenesis. Lupus-prone T-bet-deficientmice were generated by intercrossing a T-bet-deficient line with theMRL/MpJ-Fas(CD95)^(lpr/lpr) murine lupus strain, generating animals offour genotypes, T-bet^(+/+)Fas^(+/+), T-bet^(−/−)Fas^(+/+),T-bet^(+/+)Fas^(lpr/lpr)(T-bet+lpr), andT-bet^(−/−)Fas^(lpr/lpr)(T-bet-lpr). Flow cytometric analyses of tissuesfrom adult 6-week old animals revealed that T-bet did not have asignificant effect upon the proportional numbers of CD4+ or CD8+ Tcells, or B220-positive B cells in spleen or lymph node. Upon aging,T-bet-lpr animals were protected from immune-complex renal disease,which was characterized by strikingly diminished glomerular,interstitial and perivascular inflammation as well as glomerular immunecomplex deposition. Also, they developed significantly less humoralautoimmunity as assessed by the fluorescent antinuclear antibody testand two tests for anti-DNA antibodies. Their sera contained some, albeitdiminished, autoimmunity to DNA as assessed by ELISA, but were unable torecognize native, double-stranded DNA as assessed by Crithidiaimmunofluorescence, suggesting the presence of generalized (e.g.,anti-ssDNA), but not matured (e.g., anti-dsDNA) autoimmunity inT-bet-lpr animals. Compared to T-bet⁺lpr animals, T-bet⁻lpr animals wererelatively protected from glomerulonephritis-related mortality (survivalof 57%, n=7 versus 100%, n=6, at 28 weeks, respectively).

Surprisingly, T-bet⁻lpr animals continued to develop othermanifestations consistent with T-cell autoimmunity, including cutaneous,salivary gland, and hepatic infiltrates, as well as lymphoidorganomegaly, often in excess of their T-bet⁺lpr littermates. Theselymphoid infiltrates consisted mostly of T cells, as assessed byimmunohistopathology. Such findings suggest that the Th1-dominant T cellautoimmunity in this model was largely intact in the absence T-bet.Although T-bet was required for the production of IFN-γ by naive CD4⁺ Tcells from CD95-intact animals, T-bet⁻lpr T cells produced excesscytokines, including IFN-γ and IL-4, and demonstrated similarproliferative activity in an autologous mixed lymphocyte reaction,compared to their T-bet⁺lpr littermates.

Since pathogenic autoantibodies are necessary and sufficient to induceimmune complex glomerulonephritis, and T-bet is induced in both humanand murine B cells upon activation, it is likely that T-bet is directlyrequired in B lymphocyte function. As assessed by serum levels, T-betwas required for the complete expression in these lupus-prone animals ofhypergammaglobulinemia IgG2a, IgG2b and IgG3, a requirement amplified inFas^(lpr/lpr) animals IgG2a levels, however, were severely diminished inT-bet-deficient sera from either Fas genotype. IgG2a immune depositswere significantly reduced in the kidneys of T-bet-lpr animals. PurifiedT-bet-deficient B cells were unable to complete class switching to IgG2awhen stimulated in vitro, as assayed by secreted immunoglobulin. Classswitching to IgG2b and IgG3 was significantly diminished, butnevertheless present in T-bet-deficient cells. These deficits appearedto occur at the transcriptional level, since in class-switching assaysT-bet-deficient B cells were neither able to accumulate surface IgG2anor generate germline or postswitch IgG2a transcripts. Conversely,T-bet-deficient B cells produced excess amounts of the Th2-relatedisotypes IgG1 and IgE. These deficits did not simply result from anunopposed effect of IL-4, because the addition of up to 10 μg/mLanti-mIL-4 antibodies to B cell cultures did not affect the IgG2adeficiency, or the IgG1/IgE excess. These observations suggest aprofound role for T-bet in the regulation of IgG2a at the level of thegermline transcript, and further implicate it in the regulation of IgG1and IgE.

Further evidence that T-bet directly controls the transcription of IgG2aincludes the following. Transfection of the murine pre-B cell lymphoma18.81 with a T-bet expression plasmid induced endogenous IgG2a germlinetranscripts. In addition, transduction of primary T-bet-deficient Bcells with a T-bet-expressing retrovirus confers the ability to generateIgG2a germline transcripts, as well as secreted IgG2a. Furthermore,purified B cells from a CMV-T-bet transgenic mouse line, which expressesT-bet under the control of the CMV early promoter, produced increasedamounts of IgG2a when stimulated in vitro with LPS and rmIFN-γ comparedto B cells from nontransgenic littermates (490±50 ng/mL vs. 1058±120ng/mL, n=3). To determine if T-bet played a role in the IFN-γ signalingpathway, the CMV-T-bet transgenic line was crossed with an IFN-γreceptor (IFNγR)-deficient background T-bet was able to augment theproduction of IgG2a, this time in the absence of IFN-γ signaling. Theproliferative capacity of T-bet deficient B cells, as well as theirability to upregulate several markers of B cell activation, includingIFN-γ, IL-6, IL-10, and GM-CSF, was unaffected in vitro, furthersuggesting a direct role for T-bet in the regulation of IgGtranscription, independent of B cell activation status.

T-bet therefore confers upon B lymphocytes the ability to class switchto IgG2a in response to IFN-γ. T-bet also plays a significant role inthe regulation of other Ig isotypes, and thus, plays a major role in theregulation of pathogenic autoantibody production. Without being bound byone particular theory, given its role as a transcription factor, T-betlikely regulates class switching via control of germline transcripts,which have been strongly implicated as a prerequisite to isotype switchrecombination. Alternatively, T-bet may participate in mediatingaccessibility of the IgG locus to transcriptional or recombinatorialfactors, as it does for IFN-γ in CD4 T cells. In either scenario, T-betserves as a mediator of signals to transactivate the classicalIFN-γ-related immunoglobulin isotype IgG2a, yet inhibits the classicalTh2-related isotypes IgG1 and IgE. While T-bet is capable of inducinggermline transcripts in the absence of exogenous IFN-γ, completewild-type-level production of IgG2a appears to require IFN-γ signaling,suggesting that T-bet cooperates with another factor in the IFNγRpathway, such as STAT1, or at least require signaling messages, such astyrosine phosphorylation, e.g., activated by the IFNγR for completeactivity.

The identification of T-bet as a regulator of IgG isotype classswitching may prove helpful in future transcriptional analyses of thenon-IL4-dependent IgG subclasses, whose study has been greatly hinderedby their apparently very distant locus control regions. Although thepresent results demonstrate that T-bet can transactivate endogenousIgG2a transcripts in whole cells, it cannot transactivate a reporterconstruct consisting of 3 kB of putative IgG2a promoter upstream of theI exon, at least in 18.81 cells. Thus, the control region for IgG2a, atleast as it relates to T-bet, may be quite distant. The present resultsare therefore of particular significance given the complete yetselective absence of IgG2a germline transcripts in the T-bet-deficient Bcells. In comparison, several reported immunoglobulin isotypeimmunodeficiencies caused by other transcription factor knockoutsinvolve multiple Ig isotypes and/or other developmental B cell defects.Thus, the present invention identifies T-bet as an isotype-specificparticipant in the class switch mechanism.

Example 16 T-Bet Regulates Mucosal T Cell Activation in ExperimentalColitis and Crohn's Disease

Crohn's disease and ulcerative colitis are the two major forms ofinflammatory bowel diseases (IBD) in humans. Whereas Crohn's disease ischaracterized by a transmural, granulomatous inflammation that can occuranywhere in the gastrointestinal tract, ulcerative colitis causes a moresuperficial, continuous inflammation that is restricted to the largebowel. Although the etiology of the diseases is unknown, it has beensuggested that an activation of the mucosal immune system in response tobacterial antigens with consecutive pathologic cytokine production andactivation of matrix metalloproteinases plays a key pathogenic role. Inparticular, cytokines produced by T lymphocytes appear to initiate andperpetuate chronic intestinal inflammation. Interestingly, cytokineproduction by lamina propria CD4⁺ T lymphocytes differs between Crohn'sdisease and ulcerative colitis. Whereas the former disease is associatedwith increased production of T helper 1 (Th1) type cytokines such asIFN-γ and TNF, the latter disease is associated with T cells producinglarge amounts of the Th2 type cytokine IL-5 while IFN-γ production isunaffected. In both Th1- and Th2-mediated inflammatory bowel disease,the immunosuppressive cytokine TGF-β, mainly secreted by Th3 cells and aunique population of regulatory T cells (Tr), provides a powerfulprotective effect.

A. Reciprocal Expression of GATA-3 and T-Bet in Lamina Propria T Cellsfrom Patients with Crohn's Disease

Since changes in cytokine production by lamina propria T cells have beenimplicated as a key phenomenon in the pathogenesis of inflammatory boweldiseases (IBD), a series of experiments on the expression of T-bet bypurified lamina propria (LP) T cells in IBD patients was performed.Immunofluorescence double staining studies showed an accumulation ofT-bet expressing LP T cells in patients with Crohn's disease (CD). Inaddition, it was found that T-bet was strongly expressed in both thecytoplasm and the nucleus of LP mononuclear cells in patients withCrohn's disease, while only a weak staining in perinuclear areas or nostaining was observed in control patients and patients with ulcerativecolitis. To verify increased expression of T-bet in patients withCrohn's disease, nuclear extracts of purified LP T cells from patientswith Crohn's disease and control patients were isolated and expressionof T-bet by EMSA and Western blot analysis was analyzed. Patients withCrohn's disease expressed higher amounts of nuclear T-bet compared withcontrol patients.

The expression of GATA-3 in LP T cells from CD patients was alsoassessed. GATA-3 expression was downregulated in LP T cells from CDpatients compared to control patients, as assessed byimmunohistochemical double staining analysis for CD3 and GATA-3 on coloncryosections. These data are indicative of a reciprocal expressionpattern of GATA-3 and T-bet in CD LP T cells that is associated withincreased IFN-γ but decreased IL-4 and IL-5 production in this disease.

B. Induction of T-Bet Expression in Th1- but not Th2-Mediated AnimalModels of Chronic Intestinal Inflammation

Nuclear proteins from T cell enriched lamina propria mononuclear cells(LPMC) in various animal colitis models were isolated and T-betexpression was assessed by EMSA and Western blot analysis. T-bet wasfound to be strongly expressed in T cell enriched LP cells in theTH1-mediated colitis model observed in SCID or RAG mice reconstitutedwith CD62L⁺ CD4⁺ T cells. Time course studies showed that increasedT-bet expression in the colon occurred as early as 3 weeks after celltransfer before the onset of colitis. Maximum expression was noted 6weeks after cell transfer when the mice started to develop colitis,although increased T-bet expression was also observed in the full-blowncolonic inflammation seen at 12 weeks after T cell transfer.Furthermore, increased T-bet expression was consistently noted in twoadditional TH1-mediated animal models of chronic intestinalinflammation, namely colitis in IL-10 deficient mice and colitis inducedby the hapten reagent 2,4,6,-trinitrobenzene sulfonic acid (TNBS). Incontrast, unchanged or lower levels of T-bet were detected in T cellenriched LP cells in oxazolone colitis and TCRα^(−/−) μ^(−/−) associatedcolitis; two colitis models that are believed to be mediated by IL-4producing T cells and Th2 cells, respectively. These findings indicatethat T-bet is potentially a key regulator of the mucosal TH1/TH2cytokine balance in experimental colitis in vivo.

C. Retroviral or Transgenic Overexpression of T-bet Induces an EarlyOnset of Severe CD62L⁺ CD4⁺ Th1 T Cell-Mediated Colitis in SCID Mice

To determine the potential regulatory role of T-bet in Th1-mediatedcolitis in vivo by transgenic or retroviral overexpression techniques,the colitogenic potential of T cells after infection with a T-betretrovirus was analyzed. FACS-sorted GFP⁺ CD62L⁺ double positive CD4⁺ Tcells that were retrovirally transduced with T-bet induced an earlieronset of severe colitis in SCID mice compared to SCID mice reconstitutedwith control transduced CD62L⁺ T cells, as assessed by weight losscurves. This phenotype demonstrates that overexpression of T-betaccelerates development of TH1-mediated chronic intestinal inflammation.It was further observed that transfer of CD62L⁺ CD4⁺ T cells from T-bettransgenic mice induced an earlier onset of colitis activity in SCIDmice compared to T cells from wild-type littermates.

D. Mice Lacking T-Bet (T-Bet Knockout) are More Susceptible toTh2-Mediated Colitis

To determine the susceptibility of mice in which the T-bet gene has beeninactivated by homologous recombination for T cell-mediated colitis,T-bet deficient mice that exhibited an altered susceptibility toTh2-mediated colitis using the oxazolone-induced colitis model that haspreviously been shown to be dependent on IL-4 production by T cells wereanalyzed. The T-bet knockout mice showed enhanced susceptibility tooxazolone-induced colitis compared to both wild-type littermates andheterozygous T-bet mice, based on by weight curve), macroscopic andhistopathologic criteria. This was accompanied by a marked increase inIL-4 production by splenic CD3+ T cells, while IFN-γ production by thesecells was not significantly changed.

To determine whether the observed increase in IL-4 production wasresponsible for the differences between wild-type and T-bet knockoutmice, antibodies to IL-4 or control rat Ig to T-bet knockout mice afteroxazolone sensitization were administered. Antibodies to IL-4 suppressedhistologic colitis activity in oxazolone-treated T-bet knockout miceindicating that the protective role of T-bet in Th2-mediated colitis isdue to its direct or indirect regulation of IL-4 production.

E. T-Bet Deficiency Protects from Th1-Mediated Experimental Colitis inan Adoptive Transfer Model using CD62L⁺ CD4⁺ T Cells

The effects of T-bet deficiency in Th1-mediated colitis induced bytransfer of CD4⁺ CD62L⁺ CD45Rb^(high) T cells in SCID and RAG knockoutmice were assessed. Transfer of T-bet expressing CD4⁺ CD62L⁺ T cellsfrom wild-type mice resulted in clinical and endoscopic signs of severecolitis. In contrast, transfer of T-bet^(−/−) CD4⁺ CD62L⁺ T cells failedto induce chronic diarrhea, weight loss, rectal prolapse and endoscopicsigns of colitis. Furthermore, transfer of T-bet deficient T cellsresulted in a markedly reduced colitis activity in SCID micereconstituted with CD62L⁺ CD45Rb^(high) CD4⁺ T cells in threeindependent experiments, as assessed by macroscopic and histologiccriteria. This protective effect of T-bet deficiency on CD62L⁺ CD4⁺ Tcell-induced colitis was at least as pronounced as that seen upontransfer of STAT-1-deficient CD62L⁺ CD4⁺ T cells (histopathologic score:STAT-1^(−/−) reconstituted mice: 1.25+/−0.9 vs. T-bet^(−/−)reconstituted mice: 0.8+/−0.2). LP T cells from T-bet knockout T cellreconstituted mice produced less IFN-γ compared to LP cells fromwild-type T cell reconstituted mice (336+/−24 pg/ml versus 1159+/−25pg/ml) indicating that T-bet deficiency suppresses proinflammatorycytokine production by mucosal CD4⁺ T cells. Interestingly, CD4⁺ CD62L⁺T cells from heterozygous T-bet mice showed a marked variability toinduce colitis in three independent experiments, likely due to athreshold effect of T-bet expression in controlling cytokine geneexpression and hence the colitogenic potential of T cells.

F. T-Bet Controls the Mucosal Balance Between IFN-γ and IL-4 Productionby T Cell Enriched Lamina Propria Cells in the Absence of ColitogenicStimuli

The structure of the lamina propria and cytokine production by laminapropria mononuclear cells (LPMC) in mice lacking T-bet was assessed. Nomacroscopic or histologic abnormalities in the small and large bowel ofT-bet heterozygous and T-bet knockout mice in the absence of colitogenicstimuli was observed. To analyze cytokine production by T cell enrichedLPMC from T-bet deficient mice and wild-type littermates, cells werestimulated by anti-CD3 plus anti-CD28 for 48 hours and cytokineproduction in culture supernatants was determined by ELISA. Twoindependent experiments demonstrated that T cell enriched LPMC fromT-bet knockout mice secreted lower levels of IFN-γ than cells fromwild-type littermates in the absence of colitogenic stimuli. Incontrast, production of the Th2 type cytokines IL-4, IL-6 and IL-10 by Tcell enriched LPMC was augmented in T-bet deficient animals compared towild-type mice. In particular, IL-4 production by T-bet^(−/−) andT-bet^(+/−) LPMC was increased compared to T-bet expressing LPMC fromwild-type littermates (FIG. 7 b). These changes in cytokine productionwere seen using LPMC from both the small and large bowel indicating thatT-bet is a regulator of the mucosal Th1/Th2 cytokine balance in theentire gut immune system.

Whether increased Th2 cytokine production by LPMC in T-bet knockout micewas associated with evidence for activated IL-4 signaling in LP T cellswas assessed next. There was increased GATA-3 expression in nuclearextracts from T cell enriched LPMC of T-bet deficient mice, as shown byboth gel retardation assays and Western blot analysis, consistent withan increased presence of Th2 effector T cells in the mucosa of T-betknockout mice.

G. T-Bet Deficient Regulatory CD62L⁻ CD4⁺ T Cells Show EnhancedProtective Functions in Th1-Mediated Colitis and Exhibit Increased TGF-βProduction and Signaling

To determine the effect of T-bet deficiency on TGF-β production andsignaling in T cells, the T cell enriched LPMC in T-bet knockout micewere observed and shown to produce increased amounts of TGF-β comparedto cells from wild-type littermates. This increased production of TGF-βin the absence of T-bet could be important for the regulatory functionof T-bet in colitis, since TGF-β production by T cells has been recentlysuggested to play a key role in suppressing chronic intestinalinflammation. In intestinal inflammation, TGF-β is mainly produced by aunique population of regulatory CD25⁺ CD45RB^(low) CD62L⁻ CD4⁺ T cellsthat have been shown to suppress colitis activity in SCID mice whenco-transferred with CD4⁺ CD62L⁺ T cells. Furthermore, at least in thespleen, IL-4 producing T cells have been shown to produce high amountsof TGF-β in secondary cultures.

Both splenic CD62L⁺ and CD62L⁻ CD4⁺ T cells from healthy mice expressedlarge amounts of nuclear T-bet as shown by Western blot analysis.Splenic CD25⁺ cells, however, showed lower amounts of nuclear T-betexpression. Furthermore, CD62L⁻ CD45RB^(low) CD4⁺ T cells from T-betknockout mice showed decreased expression of Smad7, an inhibitory Smadprotein that is induced by IFN-γ and suppresses TGF-β signaling in Tcells, compared to cells from wild-type mice. Consistently, splenicT-bet^(−/−) CD62L⁻ CD4⁺ T cells exhibited increased nuclear Smad3expression compared to T-bet expressing CD62L⁻ CD4⁺ T cells indicativeof enhanced TGF-β signaling. Based on these findings the potentialregulatory capacity of CD62L⁻ CD4⁺ T lymphocytes from wild-type, T-betheterozygous and T-bet knockout mice to suppress colitis induced byT-bet expressing CD62L⁺ CD4⁺ T cells was analyzed. Cotransfer ofregulatory CD62L⁻ CD4⁺ T cells plus naive CD62L⁺ CD4⁺ T cells from T-betexpressing wild-type mice led to less severe colitis compared to micereconstituted with CD62L⁺ CD4⁺ T cells confirming a protective role forthis T cell subset in vivo. Moreover, cotransfer of T-bet-deficientregulatory CD62L⁻ CD4⁺ T cells caused a more pronounced protectiveeffect on CD62L⁺ CD4⁺ T cell-mediated colitis compared to regulatoryCD62L⁻ cells from wild-type mice. This finding was associated with anincreased production of TGF-β by lamina propria T cells fromreconstituted mice and by an expansion of the number of regulatory CD4⁺CD25⁺ T cells in the spleen of reconstituted mice.

H. T-Bet Controls TGF-β Production and Signaling in Regulatory T Cellsand TGF-β Inhibits T-Bet Expression

Regulatory T cells producing IL-10 or TGF-β mediate protective effectsin Th1-mediated colitis by suppressing the activity of T lymphocytes. Todetermine the if T-bet has a role in TGF-β production and signaling inregulatory T cells, the following studies were performed. Cells werecultured in the presence of antibodies to CD3 and CD28 with or withoutrecombinant IL-4 and TGF-β (1 ng/ml). Cellular extracts were made after48 hours and analyzed for the expression of T-bet and beta-actin byWestern blot analysis. To determine whether TGF-β is produced by T cellenriched LPMC from wild-type (WILD TYPE), T-bet heterozygous (HET) andT-bet knockout (KNOCK OUT) mice in the absence of colitogenic stimuli,cells were stimulated with antibodies to CD3 plus CD28 and supernatantswere analyzed by ELISA. To determine whether T-bet is expressed insplenic CD25⁺, CD62L⁺ and CD62L⁻ CD4⁺ T cells from healthy wild-typemice, cytoplasmic (CYT) and nuclear (NUC) extracts from these cells wereisolated and analyzed for T-bet expression by Western blotting. Todetermine whether TGF-β-mediated signaling is increased in T-betdeficient CD62L⁻ CD4⁺ T cells, CD62L⁻ CD4⁺ T cells from wild-type andT-bet knockout mice were stimulated with anti-CD3 plus anti-CD28 andrIFN-γ for 12 hours followed by protein extraction and Western blotanalysis. Cellular extracts were analyzed for Smad7 expression whereasnuclear extracts were analyzed for Smad3 levels. To measure theinflammation score of mice reconstituted with CD62L⁺ CD4,⁺ T cells fromwild-type mice and CD62L⁻ CD4⁺ T cells from T-bet knockout mice (KNOCKOUT) and wild-type (WILD TYPE) control mice were measured.

FACS analysis of splenic CD4+ T cells from mice reconstituted withCD62L⁺ CD4⁺ T cells from wild-type mice plus CD62L⁻ CD4⁺ T cells fromT-bet knockout mice (KNOCK OUT) or wild-type (WILD TYPE) control micewas also performed. Finally, TGF-β production by LPMC from the abovereconstituted mice was measured by stimulating cells with antibodies toCD3 plus CD28 in serum free medium. Supernatants were collected after 3days followed by analysis of supernatants by ELISA.

The foregoing studies demonstrate a regulatory role for T-bet in mucosalcytokine production. Specifically, the present invention demonstratesthat CD62L⁻ CD4⁺ T cells from T-bet knockout mice exhibit a strongerprotective effect on CD62L⁺ CD4⁺ T cell-induced colitis than thecorresponding cell population from wild-type mice. This observation isrelated to differences in TGF-β production and signaling, as CD62L⁻ CD4⁺T cells from T-bet deficient mice exhibited increased nuclear Smad3expression. After binding of TGF-β to its receptor on T cells, Smad3 isinteracts with the TGF-β receptor I followed by importin-1β andRanGTPase-mediated import of Smad3 into the nucleus where it controlsexpression of β target genes. IFN-γ has been previously shown to inhibitTGF-β signaling by a Jak1/STAT-1-mediated rapid activation of thesynthesis of the inhibitory Smad-7 protein, which in turn can preventthe interaction of Smad3 with the TGF-β type I receptor. Furthermore,Smad7 can form a complex with the ubiquitin-ligase Smurf2 that targetsthe TGF-β receptor for degradation. Thus, the reduced production ofIFN-γ by splenic CD4⁺ T cells and T cell enriched lamina propria cellsin T-bet deficient animals causes reduced expression of Smad7 followedby increased TGF-β signaling via Smad3/4. In fact, CD62L⁻ CD4⁺ T cellsfrom T-bet deficient mice express reduced levels of Smad7 compared to Tcells from wild-type mice.

Since administration of neutralizing antibodies to TGF-β is known tosuppress the protective capacity of CD62L⁻ CD4⁺ T cells on Th1-mediatedcolitis, the present invention demonstrates that the enhanced regulatorycapacity of T-bet deficient CD62L⁻ CD4⁺ cells is due to increased TGF-βproduction and signaling. The enhanced TGF-β production of T-betdeficient T cells is likely augmented after T cell transfer, as TGF-βhas been demonstrated to positively regulate its own production. Therelevance of a defect in TGF-β1 expression or TGF-β-mediated signalingvia Smad3 for the mucosal immune system has been shown by theobservation that knockout mice for these proteins develop Tcell-mediated chronic intestinal inflammation. TGF-β in turndownregulates T-bet expression in mucosal cells, demonstrating areciprocal relationship between TGF-β and T-bet levels in T cells. Weobserved that treatment of activated T-cell enriched LPMCs with TGF-βbut not IL-4 suppressed T-bet expression suggesting a reciprocalrelationship between TGF-β and T-bet (FIG. 13).

In summary, the present invention identifies T-bet as a master switchfor T cell-mediated chronic intestinal inflammation and the regulationof protective immune responses by TGF-β. T-bet controls Th1 and Th2cytokine production in colitis and its levels are downregulated byTGF-β. Furthermore, downregulation of T-bet is associated with increasedTGF-β levels due to failure of T-bet-mediated activation of Smad7.Moreover, TGF-β levels correlate with colitis in mice lacking T-bet.Thus, modulation of T-bet function is a valuable target for localtherapeutic intervention in Th1-mediated chronic intestinal inflammationsuch as is observed in Crohn's disease.

Example 17 Mice Lacking T-Bet Spontaneously Develop Airway ChangesConsistent with Human Asthma

Human asthma is associated with reversible airway obstruction, airwayinflammation, airway hyperresponsiveness (AHR) and, in chronic asthma,airway remodeling. Murine models of asthma mimic many of the features ofthe human disease. In these models the production of IL4, IL-5 and IL-13have been associated with the development of an asthma-like phenotype.In an adoptive transfer model, enhanced expression of IFNγ by Th1 cellsin the airway protects against allergic disease, but the presence of Th1cells does not attenuate Th2 cell-induced airway hyperreactivity andinflammation.

To determine whether T-bet was expressed in the lungs of normalindividuals and in patients with allergic asthma, immunohistochemistryusing a mAb to T-bet was performed. The results revealed expression ofT-bet in thirteen normal control lungs, but very little expression inseven patients with allergic asthma. Double staining for CD4 and T-betin consecutive sections showed that most of the cells expressing T-betwere CD4⁺ T cells. Thus, T-bet deficiency recapitulates many aspects ofthe asthmatic phenotype.

Naïve mice, i.e., neither antigen sensitized nor challenged, with atargeted deletion of T-bet were examined to ascertain if such animalswould manifest various aspects of the induced asthma phenotype. Comparedto wild type (wild type) mice, those either heterozygous (T-bet+/−) orhomozygous for a targeted deletion of T-bet (T-bet−/−) exhibited greaterairway responsiveness, as measured in unanesthetized animals by theenhanced pause response (Penh), following aerosol exposure tomethacholine. These findings were confirmed in mice that had beensensitized by systemic exposure to ovalbumin but sham challenged withaerosol phosphate buffered saline (termed OVA/PBS), by the demonstrationthat both T-bet (+/−) and (−/−) mice manifested airwayhyper-responsiveness, as compared to wild type mice, when the pulmonaryresistance response resulting from intravenous infusion of methacholine,was used as the outcome indicator. Histopathologic analysis of theairways of T-bet (−/−) mice at baseline demonstrated peribronchial andperivenular infiltration with eosinophils and lymphocytes as compared tocontrol wild type litermates. T-bet deficient mice had increaseddeposition of fibroblast-like cells beneath the basement membrane.Eosinophils were not present in the bronchoalveolar lavage fluid ofT-bet deficient mice despite enhanced recovery of IL-5. T-bet+/−heterozygous mice, that have only a 50% reduction in T-bet protein,displayed a phenotype very similar to mice with a complete absence ofT-bet.

In contrast to the spontaneous asthma observed in T-bet−/− and +/−animals, many murine models of asthma, depend on a protocol of primingand sensitization to allergen to elicit disease. Thus, OVA aerosolchallenge of mice previously sensitized to OVA was tested to determinewhether it would lead to enhanced airway responsiveness in T-betdeficient mice as it does in wild type mice. The pulmonary resistanceresponse observed after intravenous infusion of methacholine in T-betdeficient mice sensitized to OVA and receiving aerosol challenge wassimilar to that observed in mice who did not receive aerosol OVAchallenge. After OVA/OVA exposure, no differences were observed betweenwild type or T-bet deficient mice with respect to the infiltration ofthe airways with eosinophils or lymphocytes or in the cellularcomposition of the bronchoalveolar lavage fluid. Thus, mice with reducedor absent levels of T-bet display a spontaneous, non-allergen-inducedasthma phenotype that is not further exacerbated with antigenicstimulation.

The thickness of the sub-epithelial collagen layer was evaluated in wildtype and T-bet−/− and +/− deficient animals. In the wild type animalsthere was minimal deposition of collagen beneath the basement membrane,while in the T-bet deficient mice the sub-basement membrane collagenlayer was significantly thicker than it was in wild type mice. Inaddition to a thickened collagen layer, there were increased numbers ofbronchial myofibroblasts, as assessed by immunostaining for alpha-smoothmuscle actin. These data indicate that the airways of T-bet deficientmice undergo remodeling similar to that observed in humans with chronicasthma.

Whether these structural changes in the airway were associated with adifference in the patterns of cytokine expression between wild type andT-bet deficient mice was examined TGF-beta, a potent stimulator oftissue fibrosis, TNFα and Il-4, another pro-inflammatory cytokineimplicated in the chronic remodeling of the airways in asthma, wererecovered in increased amounts from the BALF of mice homozygous for theT-bet targeted deletion. T-bet deficiency induced a selective alterationin patterns of cytokine expression as no significant changes wereobserved in IL-10 and IL-6 production. Although the physiological andhistologic findings were similar in mice either hetero- or homozygousfor the targeted deletion, only the homozygous mice exhibited anincreased production of cytokines.

The identity of the cells in T-bet deficient mice responsible for airwayhyperreactivity and airway inflammation was examined by adoptivetransfer of spleen CD4+ cells from different groups of OVA sensitizedmice into histocompatible SCID mice. To enhance the localization of thetransferred T cells into the lungs of mice, the OVA aerosol wasadministered one day before adoptive transfer of the T cells. On the dayfollowing the adoptive transfer, OVA aerosol exposures were begun andcontinued for three days. Four days after cell transfer, lung mechanicswere evaluated. Control SCID mice received an intraperitoneal infusionof saline rather than T cells suspended in saline. Recipients of wildtype spleen CD4 cells had comparable airway responsiveness to wild typemice that received OVA sensitization but were not challenged. Mice thathad been reconstituted with CD4 cells lacking T-bet showed increasedairway hyperresponsiveness as compared to mice reconstituted with CD4cells derived from wild type littermates and similar to that of OVAsensitized mice lacking T-bet. CD4 staining of BALF cells harvestedafter measurement of lung mechanics was performed to assure that CD4⁺cells were recruited to the lung; the proportion of lymphocytes thatwere CD4 positive in wild type (+/+) mice was 38.9%+/−2.2; in CD4 T-bet(+/−) mice was 39.57%+/−6.48; and in T-bet (−/−) mice was 38.5%+/−5.48.In addition, the lungs of SCID mice reconstituted with CD4 cells derivedfrom T-bet (−/−) mice exhibited increased IL-4, IL-5 and IL-13 in theBALF as compared to recipient mice reconstituted with spleen CD4+ cellsderived from wild type mice, demonstrating that the airwayhyperreactivity observed in T-bet (−/−) mice is T-cell mediated.Furthermore, treatment of T-bet knock out mice with antibodies to IL-13abrogated airway hyperreactivity indicating that one mechanism by whichT-bet acts is to regulate the production of IL-13.

The present invention demonstrates that targeted deletion of T-bet, inthe absence of an induced inflammatory response, results in aphysiological and inflammatory phenotype in murine airways similar tothat created by allergen exposure in sensitized mice. In addition toacute inflammatory changes, T-bet deficient mice demonstrate airwayremodeling consistent with asthma that is reminiscent of the humandisease. Remarkably, this phenotype exists spontaneously and isfull-blown, since, when sensitized and challenged with allergen, T-betdeficient mice fail to enhance either their physiological or pathologicresponses.

Example 18 Mice Lacking T-Bet are Resistant to EAE Development

Mice lacking T-bet were evaluated in a murine model of EAE. Myelinoligodendrocyte glycoprotein (MOG) is able to generate anencephalitogenic T cell response in normal animals in the presence ofpertussis toxin. T-bet^(−/−) and T-bet^(−/+) mice were immunized with apeptide derived from myelin oligodendrocyte glycoprotein (MOG 35-55) toinduce EAE. Groups of mice were followed for up to 25 days to score forclinical disease as described (Bettelli et al., 1998. J. Immunol. 161:3299-3306). Briefly, mice are injected s.c. in the flank with anemulsion containing 200 g of the peptide MOG₃₅₋₅₅, which is theencephalitogenic epitope in C57BL/6 (H-2^(b)) mice (24) and CFAsupplemented with 400 ug of Mycobacterium tuberculosis H37 Ra (DifcoLaboratories). Mice were observed daily and assessed for clinical signsof disease according to the following criteria: 0, no disease; 1, limptail; 2, hind leg weakness or partial paralysis; 3, complete hind legparalysis; 4, front and hind limb paralysis; 5, moribund state. Meanclinical score is calculated as follows: individual scores are added anddivided by the total number of mice in each group for each day ofobservation; this includes the animals that do not develop any disease.Animals were sacrificed at the termination of the experiment or at thepeak of disease. As shown in FIG. 14, T-bet^(−/+) mice suffered fromsevere paralysis, the mean clinical score for this group of animals wasapproximately 3 at day 45. In contrast, T-bet^(−/−) mice did not developparalysis; this group of animals had a mean clinical score of less than1.

2D2 MOG specific T cell receptor (TCR) transgenic mice were mated withT-bet^(+/+) and T-bet^(−/−) mice. Mice from each of the groups wereimmunized with pertussis toxin to induce EAE. As shown in FIG. 15, theanimals lacking T-bet had a mean clinical score of less than 0.5 andwere protected from EAE in this model. The role of another transcriptionfactor acting upstream of T-bet was also tested in this model of EAE.Most IFN-γ responses are coupled to the Jak-Stat signaling pathway, inparticular to the protein tyrosine kinases Jak1 and Jak2 and thetranscription factor Stat1.

The proliferation of CD4⁺ cells from these mice was also examined. CD4⁺cells were cultured with 0, 1, 10, or 100 ug/ml of MOG 35-55 peptide inthe presence of irradiated spleen cells as antigen presenting cellsusing standard methods. ³H-thymidine uptake was used to measureproliferation of the cells. IFN-γ production by CD4 cells was measuredby intracellular staining (Szabo et al. Cell. (2000 Mar. 17)100(6):655-69).

FIG. 16 shows that the percentage of CD4⁺ cells staining positive forIFN-γ was 33% in 2D2 MOG×T-bet^(+/+) animals and 3% in 2D2MOG×T-bet^(−/−) animals.

Example 19 Mice Lacking T-Bet Demonstrate Attenuated Arthritis

Rheumatoid arthritis (RA) is a form of arthritis in which the membranesor tissues lining the joints become inflamed (synovitis). Jointinflammation causes swelling and pain and, over time, may destroy thejoint tissues and lead to disability. RA affects the hands, wrists,elbows, feet, ankles, knees, or neck. It usually affects both sides ofthe body at the same time. In unusual cases, rheumatoid arthritis mayaffect the eyes, lungs, heart, nerves, or blood vessels. Late stage RAcan cause boutonniere deformity of the thumb, ulnar deviation ofmetacarpophalangeal joints and swan-neck deformity of fingers. Todetermine whether T-bet deficiency effects the development of RA, ananimal model of RA was employed. 6-8 week old female Balb/c control andT-bet^(−/−) mice were injected with a anti-type II collagen monoclonalantibody cocktail (Terato et al Journal of Immunology 148, 2103-2108,1992; Kagari et al, Journal of Immunology. 169:1459) by tail veininjection to induce Collagen Antibody-Induced Arthritis (CAIA). The micewere given an LPS injection (50 to 100 micrograms) intraperitoneally 72hours later and the development of arthritis was evaluated on days 5 and15. The bet deficient mice demonstrated attenuated arthritis compared tothe wild-type controls.

Example 20 T-Bet Regulates the Generation of CD8 Effector/Memory Cells

To assess the role of T-bet in CD8 cell function, T-bet^(−/−) mice weremated to the TCR transgenic mouse OT-1, which recognizes the ovalbuminpeptide 257-264 in the context of K^(b). In these mice virtually all Tcells are CD8 cells and are specific for one peptide. A substantialdecrease (approximately two thirds reduction) in numbers of CD8 cells inthe absence of T-bet was observed. Impaired generation of the effectorCD8 population was observed in T-bet^(−/−) mice as evidenced by thereduction in CD8⁺, CD44Hi, CD62Lhi, CD69Hi and Ly6CHi cells. FIG. 17shows the results of FACS analysis performed on cells from wild-type andT-bet^(−/−) mice.

To determine if T-bet transcripts were expressed and induced in CD8 Tcells as in CD4 T cells, RNA was isolated from purified CD8 cellsactivated for 72 hrs with plate-bound anti-CD3 and anti-CD28 in thepresence or absence of IL-12 and IL-18 (Szabo et al. Cell. (2000 Mar.17) 100(6):655-69). Northern blot analysis showed the coordinateinduction of both T-bet and IFNγ RNA indicating that T-bet expressioncorrelated with IFNγ production in CD8 cells. IFNγ production fromcytotoxic CD8 cells is a key mechanism by which these cells combat viralinfections; effector and memory CD8 cells produce large amounts of IFNγ.

To determine whether IFN-γ production was affected in T-bet deficientCD8 cells, CD8 T cells were purified from the lymph nodes ofT-bet^(−/−), T-bet^(+/−) and T-bet^(+/+) mice and stimulated withplate-bound anti-CD3 and anti-CD28 for 7 days. On day 7 these cells wererestimulated either with plate bound anti-CD3 for 24 hrs orPMA/ionomycin for 5 hours and IFNγ production was measured by ELISA orintracellular cytokine staining (ICCS). No difference in the level ofIFNγ produced or in the number of IFNγ producing cells between the threegenotypes was found. Thus, although retroviral transduction of T-betinto CD8⁺ Tc2 cells was found to convert them into Tc1 cells these datasuggested that T-bet was not required for IFNγ gene transcription inthese cells.

However, in subsequent experiments using a more physiological stimulus,the absence of T-bet was found to reduce IFN-γ production in CD8+ cells.Upon stimulation with antigen plus APCs, OT-1 TCR transgenic CD8+ cellsproduce IFN-γ. In the T-bet^(−/−) animals, however, IFN-γ production wasalmost completely eliminated (FIG. 18). Therefore, T-bet is required forIFN-γ production in CD8+ cells.

Naïve, effector and memory CD8 cells can be distinguished phenotypicallyby the expression of four markers, CD25, CD62L, CD69 and Ly-6C. Twocytokines, IL-7 and IL-15, control the generation and homeostasis of CD8memory cells. Thus, IL-15 and IL-15R knock out mice have reduced numbersof CD8 cells and virtually lack CD44hi memory CD8 cells. In the absenceof IL-15, IL-7 can maintain a memory response. However, thetranscription factors that determine the fate of a CD8 cell, i.e., itsresponse to extracellular stimuli such as cytokines, are unknown.

To search for a function for T-bet in CD8 cells, two different models ofCD8 function were examined. In the first model, the role of T-bet inhandling infection with lymphocytic choriomeningitis virus LCMV wasexamined. T-bet wild-type (WILD TYPE) or knock-out (KNOCK OUT) mice wereinfected with LCMV. At day 7 of the primary response, Tbet^(−/−) LCMVCD8 cells were isolated by Class I tetramer staining (gp120 antigen),and found to be present at equal numbers by tetramer staining (Altman etal. 1996. Science 274:94; McHeyzer-Williams et al. 1996. Immunol. Rev.150:21) and unimpaired functionally as assessed by CTL lysis ofLCMV-infected target cells and IFNγ production. However, by day 14,Tbet^(−/−) mice had a decrease in number of LCMV specific CD8 cells asmeasured by both tetramer staining and by intracellular cytokinestaining for IFN-γ.

These results indicate an impaired CD8 memory response to LCMV in theabsence of T-bet. In the LCMV model systems, a careful time course ofthe effector/memory response starting at day 8 and monitoring phenotypicmarkers, cell number, ICCS for cytokines and CTL activity must beperformed. One possibility for the results obtained is that the lack ofCD8 effector/memory could be a result of the deficiency of Th1 CD4 cellsproducing IL-2 and IFN-γ. Although MHC class II deficient mice have anormal CD8 LCMV CTL response, CD4 cells are important in LCMV infectionin the immunocompetent host. In addition, T-bet actually represses IL-2production, and CD8 cells from Tbet^(−/−) mice produce substantiallymore IL-2 than control CD8 cells.

In the second model, the T-bet knock out strain was mated to micetransgenic for the OT-1 TcR. In comparing the number and cytokineprofile of OT1 transgenic CD8 cells from wild type and T-bet knock outmice, a substantial decrease in numbers of CD8 cells in the absence ofT-bet was found, as well as a greatly increased production of IL-10 inresponse to the OVA peptide plus APC in vitro. Examining the phenotypeof CD8 cells in this model has shown a substantial decrease in thegeneration of effector CD8 cells in the absence of T-bet and a dramaticincrease in the levels of IL-10. In addition, the expression of theIL-15 receptor is not altered in freshly isolated OT1 CD8 T-bet^(−/−)cells.

Example 21 T-Bet Represses the Production of IL-10 in Effector CD8 Cells

One important function for T-bet in CD8 cells appears to be the controlof the production of the immunosuppressive cytokine IL-10. CD8 cellsthat lack T-bet were found to produce substantially increased levels ofIL-1 and IL-2 suggesting that T-bet is a repressor of IL-10 and IL-2 inCD8 cells (FIG. 36). IL-10 plays a key role in hampering the effectivehandling of intracellular pathogens such Mycobacterium, Francisellatularensis and Salmonella typhimurium because it opposes themacrophage-activating functions of IFN-γ and TNFα. IL-10 antagonizesIL-12 mediated protection against acute vaccinia virus infection andsuppression of endogenous IL-10 in dendritic cells enhances antigenpresentation for Th1 induction. Thus, compounds that enhance T-betexpression or activity should both increase IFN-γ and inhibit IL-10production, tipping the balance in favor of a potent immune response topathogens. The mechanism by which T-bet represses IL-10 or IL-2production may be by direct regulation of the transcription of the IL-10or the IL-2 gene or by regulation of genes that modulate the productionof IL-10 or IL-2 in CD8 cells.

Example 22 T-Bet Regulates the Production of IFN-γ in NK Cells

T-bet also regulates IFN-γ production in cells of the innate immunesystem. For example, IFN-γ production is reduced in animals deficient inT-bet. DX5⁺ splenic NK cells were purified from T-bet^(−/−), T-bet^(+/−)and T-bet^(+/+) mice by positive selection with NACS purification (Szaboet al. 2002. Science. 295:338-42). IFN-γ production was measured byELISA 72 hours after treatment with IL-12 alone or rIL-12 and rIL-18.Intracellular cytokine staining for IFN-γ was also performed. FIG. 19shows that IFN-γ production was reduced in both T-bet^(−/+) andT-bet^(−/−) animals.

In addition to these effects on IFN-γ production, the generation of NKcells is also impaired in the absence of T-bet. The percentage ofsplenic NK cells in WILD TYPE and T-bet^(−/−) animals were compared. Thepercentage of DX5⁺ NK cells was reduced by 50% in the absence of T-bet(FIG. 20). In another experiment, the percentage of DX5⁺ NK cells wasexamined in RAG2^(−/−) animals and Rag2^(−/−)×T-bet^(−/−) animals. RAG2is necessary for efficient V-to-DJ rearrangement during T and B celldevelopment, therefore, RAG2^(−/−) animals are deficient in T and Bcells. The percentage of DX5⁺ NK cells was also decreased in theRag2^(−/−)×T-bet^(−/−) animals as compared to the RAG2^(−/−) animals(FIG. 20).

NK cytolytic activity was also examined. Diminished spontaneous tumorcell lysis was observed in T-bet^(−/−) NK cells. Unfractionatedsplenocytes were incubated for 4 hours with 51 Cr-labeled NK-sensitiveYAC-1 target cells at the effector-to-target cell ratios indicated inleft panel of FIG. 21. (Szabo et al. 2002. Science. 295:338-42). In theright panel, T-bet^(−/−) and T-bet^(+/+) mice were injectedintraperitoneally with 100 ug of poly (I:C) 24 hours before splenocyteisolation. In another experiment, the effect of T-bet on the expressionof lytic genes was assessed. DX5⁺ NK cells were isolated from thespleens of T-bet^(+/+) and T-bet^(−/−) animals and incubated in thepresence of rIL-12 and rIL-18 for 6 hours. The abundance of mRNAencoding the lytic proteins perforin and granzyme B was determined usingBeta-actin as a reference. Expression of these lytic genes was impairedin the absence of T-bet (FIG. 22).

Example 23 T-Bet Regulates the Production of IFN-γ in Dendritic Cells

Dendritic cells (DCs) are professional antigen presenting cells with anextraordinary capacity to activate naive T cells (Liu, Y. J., et al.(2001) Nat Immunol. 2:585; Mellman, I., et al. (2001) Cell 106:255; andBanchereau, J., et al. (2000) Ann. Rev. Immunol. 18:767). They arewidely distributed in the lymphoid and non-lymphoid systems and muchinterest has been stimulated by their potent capacity to capture antigenin the lymph nodes and present it as diverse peptides to CD4 and CD8cells to initiate a primary immune response. They have indeed beendubbed “Nature's adjuvants” and must be considered to be a vitalcomponent of any vaccination strategy since current vaccine strategiesoften generate weak immunity. When triggered by pathogens, thepattern-recognition receptors (Toll receptors) expressed on immature DCsleads them to mature to immunogenic DCs. Both human and mouse DCs aredivided into subsets that subsume different functions in driving theexpansion of T helper subsets. One mechanism by which DCs do this is thesecretion of cytokines such as IL-12 and IL-10.

It has recently been discovered that dendritic cells also produce IFN-γ(Frucht, D. M., et al. (2001) Trends Immunol 22:556; Ohteki, T., et al.(1999) J. Exp. Med. 189:1981; Fukao, T., et al. (2000) Eur J Immunol30:1453; Hochrein, H., et al. (2001) J Immunol 166:5448; Fukao, T., etal. (2001) J Immunol 166:4446; Fukao, T., et al. (2000) J Immunol164:64; and Stober, D., et al. (2001) J Immunol 167:957). IFN-γ is apleiotropic cytokine essential for both innate and adaptive immunitythat acts by binding to a widely expressed IFN-γ receptor (Bach, E. A.,et al. (1997) Annu Rev Immunol 15:563; Boehm, U., (1997) Annu RevImmunol 15:749). Most IFN-γ responses are coupled to the Jak-Statsignaling pathway, in particular to the protein tyrosine kinases Jak1and Jak2 and the transcription factor Stat1 (Leonard, W. J., et al.(1998) Ann. Rev. Immunol. 16:293.). Analysis of mice lacking IFN-γ, theIFN-γ receptor, or Stat1 reveals a profound disruption of both innateand adaptive immunity resulting in death from infection by microbialpathogens and viruses (Decker, T., (2002) J Clin Invest 109:1271;Dalton, D. K., (1993) Science 259:1739; Durbin, J. E., (1996) Cell84:443; Huang, S., (1993) Science 259:1742; Meraz, M. A., (1996) Cell84:431). In mice lacking a functional IFN-γ gene, disseminatedtuberculosis occurred. Humans with inactivating mutations in componentsof the IFN-γ signaling pathway die at an early age from uncontrolledmycobacterial infections.

The cells that produce IFN-γ and respond to it reside in both the innateand adaptive immune systems. For example, this effector cytokineactivates macrophages and renders dendritic cells more immunogenic,likely by upregulating MHC antigens, antigen-presenting capacity andcytokine secretion (Giacomini, E., et al. (2001) J Immunol 166:7033;Remoli, M. E., et al. (2002) J Immunol 169:366; Kinjo, Y., et al. (2002)J Immunol 169:323; McShane, H., et al. (2002) Infect Immun 70:1623).Uncovering the molecular mechanisms that regulate IFN-γ secretion at thesites of infection and of antigen presentation is thus an importanttask. To date, Stat4 is the only transcription factor known to controlthe production of IFN-γ in myeloid cells (Fukao, T., et al. (2001) JImmunol 166:4446; Frucht, D. M., et al. (2000) J. of Immuno. 164:4659).

To determine whether T-bet is expressed in dendritic cells at levelscomparable to Th1 cells and is necessary for the optimal production ofIFN-γ, the following studies were performed:

1. Normal Development and Activation of Murine Dendritic Cells in MiceLacking T-Bet

Very few transcription factors have been implicated in the developmentand maturation of DCs (Ouaaz, F., et al. (2002) Immunity 16:257). Mostnotable among these are members of the NFκB family. Recent studies havedemonstrated impaired development of both CD11c⁺CD8a⁺ and CD11c⁺CD8a⁻DCsin mice lacking both the RelA (p65) and p50 NFκB subunits. However,macrophage development and differentiation was unaffected in these mice,proving that the requirement for p50 and RelA in generation of DCs isspecific and cell-autonomous (Ouaaz, F., et al. (2002) Immunity 16:257).Since T-bet is expressed in stem cells and progenitor cells from humanbone-marrow and umbilical cord blood, it was possible that it might alsobe involved in the development, differentiation or activation of DCs.

DCs of myeloid origin can be most easily obtained by culturingbone-marrow (BM) in the presence of GM-CSF and stages of development canbe monitored over time by assessing surface expression of MHC class IImolecules and CD11c (Lutz, M. B., et al. (1999) J Immunol Methods223:77: Inaba, K., et al. (1992) J Exp Med 176:1693). Precursor DCs(CD11c⁺MHC II^(lo)) are apparent at day 4, immature DCs (CD11c^(hi) MHCII⁺) days 4 to 8, and mature DCs (CD11c^(hi) MHC II^(hi)), days 8 to 10followed by apoptosis. There was no significant difference in the yieldof DCs at any stage of development in mice lacking T-bet, and similarproportions of precursor, immature and mature DCs, as determined bysurface phenotype were obtained from T-bet^(−/−) and control BM (day 8shown in FIG. 23A).

BM cultures containing GM-CSF provide a large population of CD11c⁺ andCD11b⁺ DCs that are derived from myeloid precursors. However, thispopulation lacks the DC subpopulations that usually reside in thesecondary lymphoid organs and are characterized by the surfaceexpression of CD4⁺ and CD8α⁺ markers (Wu, L., et al. (1996) J Exp Med184:903; Vremec, D., et al. (1997) J Immunol 159:565; Leenen, P. J., etal. (1998) J Immunol 160:2166; Kamath, A. T., et al. (2000) J Immunol165:6762; Henri, S., et al. (2001) J Immunol 167:741. 20; Hochrein, H.,et al. (2001) J Immunol 166:5448). We examined these DC subpopulationsin spleen preparations from wild type and T-bet^(−/−) mice. FACSanalysis revealed no obvious difference in DC composition based onstaining with antibodies to CD11c⁺ and a combination of I-A^(b) (MHCclass II), CD11b⁺, CD4⁺, and CD8α⁺ (FIG. 23B). Similar numbers of CD11c⁺ DCs were isolated from multiple independent preparations of spleenfrom wild and knock-out animals.

Microbial stimuli, proinflammatory cytokines, and interaction withCD40L-expressing T cells, can induce the maturation of DCs. Mature DCsare characterized by up-regulation of MHCII, co-stimulatory, andadhesion molecule expression. In order to test whether T-bet plays arole in DC maturation in vivo, mice were injected intraperitoneally withLPS for different time periods and DC maturation assessed by FACSanalysis. Up-regulation of CD86 (B7-2) in T-bet^(−/−)DCs was normalcompared to cells from wild-type mice (FIG. 23C). Similar results wereobtained for the up-regulation of CD80 (B7-1), CD40, and MHC II.

These studies show that T-bet does not play a noticeable role in thedevelopment, differentiation or activation of DCs.

2. T-Bet Expression in Murine Dendritic Cells

T-bet expression is almost exclusively restricted to the hematopoieticsystem during mouse development with the only exception being theolfactory bulb (Faedo, A., et al. (2002) Mech Dev 116:157). T-bet isexpressed in several blood lineages including progenitors/stem cellsfound in human bone marrow and cord blood (Faedo, A., et al. (2002) MechDev 116:157). In the adult animal, expression of T-bet is primarilyevident in lymphoid organs. T-bet is also expressed in Th1 cells, CD8cells, NK cells and B cells, and others have noted T-bet expression inhuman monocytes and myeloid DCs.

To examine the constitutive and regulated T-bet expression in mousedendritic cells and macrophages, immature DCs of myeloid origin wereobtained from BM cells cultured in the presence of GM-CSF. Suchbone-marrow cultures typically yield DCs at a purity range between 70 to90%. To further purify away DCs from contaminating macrophages ormyeloid precursors, CD11c⁺ magnetic beads were used resulting in apopulation of >than 95% DCs. No T-bet mRNA was detected in unstimulatedBM cells, or from cells isolated with CD11c⁺ magnetic beads at differentdays during development until day 8. At day 8, CD11c⁺ BM-derived DCscells displayed a rapid up-regulation of T-bet mRNA after stimulationwith IFN-γ (FIG. 23B). A comparable expression pattern for T-bet wasobtained in BM-derived DCs obtained from the B6 strain and from theBALB/c strain.

In order to obtain pure populations of splenic DCs, spleen cells fromC57B1/6 mice were FACS sorted with antibodies to CD11c⁺ and I-A^(b) (MHCclass II) to avoid contamination with CD8 T cells and NK cells. Suchsorted populations were >95% pure. Sorted DCs were cultured for varioustime periods in the presence or absence of recombinant IFN-γ, and RNAisolated for real-time PCR analysis. These experiments revealed lowlevels of T-bet expression in unstimulated DCs and a rapid up-regulationof T-bet transcript levels after treatment with IFN-γ (FIG. 24A).Similar to BM-derived DCs, T-bet mRNA expression peaked during the firsthour, remained high up to 4 hours, and declined dramatically after 8hours (FIG. 24B). Of note, the levels of T-bet transcripts in dendriticcells were very comparable to what has been observed in Th1 cellsranging between 10⁻³ to 10⁻² molecules of T-bet per 1 molecule ofβ-actin (FIG. 24A-B)

The expression of T-bet protein as assessed by western blot analysis ofextracts prepared from splenic DCs untreated or treated with IFN-γmirrored RNA expression with a rapid induction of T-bet proteinbeginning at 3 hours, peaking at 12 hours, and decreasing 24 hours afterstimulation with IFN-γ (FIG. 24C). No difference was noted in T-betexpression between B6 and BALB/c strains. Three-color FACS sortingallowed the examination of T-bet expression in CD8α⁺ and CD8α⁻DCsubpopulations. Both subsets up-regulated T-bet mRNA levels similarlyafter stimulation with IFN-γ. Other stimuli known to activate dendriticcells (e.g., LPS) or recently described to increase T-bet expression inNK cells (e.g., IL21 and IL15) did not induce T-bet expression.

These studies demonstrate that dendritic cells express T-bet and thatthis expression is controlled by IFN-γ in a positive feedback loopsimilar to what has been observed in T cells. These data confirm earlierobservations on the ability of IFN-γ to control T-bet expression inhuman monocytes and myeloid dendritic cells and extend it to mousemyeloid DCs at different stages of development as well as to mature DCsfrom mouse spleen (Lighvani, A. A., et al. (2001) Proc. Natl. Acad. Sci.USA 98:15137). However, some important differences between speciesemerged. For example, we did not observe significant expression levelsof T-bet mRNA or protein at 24 hours as reported for human myeloidDCs—the kinetics of T-bet induction being more rapid in mouse myeloidand splenic DCs. In addition, exquisite sensitivity to low dose IFN-γwas observed with maximal levels of T-bet obtained at 1 ng/ml. This isin contrast to human monocytes where T-bet mRNA levels increasedproportionally with higher concentrations of IFN-γ (Lighvani, A. A., etal. (2001) Proc. Natl. Acad. Sci. USA 98:15137). Furthermore, T-betexpression was not detected in either peritoneal, splenic or bone marrowderived macrophages upon treatment with IFN-γ, or after phagocytosis oflatex beads. These studies demonstrate that the production of IFN-γ fromthese cells is controlled by transcription factors other than T-bet suchas Stat4 in macrophages of the BALB/c background (Frucht, D. M., et al.(2000) J. of Immuno. 164:4659; Lawless, V. A., et al. (2000) J. Immunol.165:6803; Kuroda, E., et al. (2002) J. Immunol. 168:5477).

3. T-bet is Essential for Optimal Production of IFN-γ by Dendritic Cells

Upon a 72 hour stimulation with IL12 and IL18, DCs secrete substantialamounts of IFN-γ ranging between 10 to 300 ng ml⁻¹ (Ohteki, T., et al.(1999) J. Exp. Med. 189:1981; Fukao, T., et al. (2000) Eur J Immunol30:1453; Hochrein, H., et al. (2001) J Immunol 166:5448; Fukao, T., etal. (2001) J Immunol 166:4446; Fukao, T., et al. (2000) J Immunol164:64; Stober, D., et al. (2001) J Immunol 167:957). To date, Stat4 isthe only transcription factor known to control the production of IFN-γin myeloid cells. In both DCs and macrophages, the IL12-dependentsecretion of IFN-γ is severely diminished in the absence of Stat4 (Bach,E. A., et al. (1997) Annu Rev Immunol 15:563). In addition, Stat4^(−/−)macrophages exhibited defective production of nitrate oxide in responseto IL-12, and are susceptible to Toxoplasma gondii infection (Fukao, T.,et al. (2001) J Immunol 166:4446). Since T-bet controls thetranscription of the IFN-γ gene in CD4⁺ T cells, but not, for example inB cells, whether T-bet controls the transcription of IFN-γ in DCs wasstudied.

A very marked impairment in IFN-γ secretion was observed in BM DCsderived from T-bet deficient B6 mice. Although IFN-γ production by BMderived DCs is typically lower than from splenic DCs, ranging from 100to 5000 pg ml⁻¹ (Fukao, T., et al. (2001) J Immunol 166:4446; Fukao, T.,et al. (2000) J Immunol 164:64; Stober, D., et al. (2001) J Immunol167:957), T-bet^(−/−) BM DCs produced no detectable IFN-γ at all asmeasured by ELISA (FIG. 25A, left panel). BM-derived wild type B6 DCsproduced levels that ranged from 100 to 500 pg ml⁻¹ (FIG. 25A, leftpanel). Unlike splenic DCs which die in culture after 48 to 72 h,BM-derived DCs survive for long enough periods of time to allow us tomeasure IFN-γ transcripts as well. Survival rates were similar inBM-derived DCs from T-bet and wild-type mice after stimulation for 72hours. Real-time PCR analysis confirmed a very marked decrease (6 to 12fold) in IFN-γ transcripts in T-bet^(−/−) BM-derived DCs cultured withIL-12 and IL-18 for 72 hours (FIG. 25A, right panel). Comparable resultswere obtained in BM derived DCs from T-bet^(−/−) mice on a BALB/cbackground.

Similarly, a significant reduction in IFN-γ secretion, ranging from 40to 80% from six independent preparations of splenic DCs stimulated withIL-12, or IL-12 and IL-18, was observed in T-bet deficient as comparedto control mice (FIG. 25A). Similar results were observed in DCs derivedfrom T-bet^(−/−) mice on the BALB/c background. T-bet functions veryearly in Th differentiation in the naïve Th progenitor cell to regulateIFN-γ gene transcription. Optimal production of IFN-γ by murinedendritic cells has been reported to occur between 3 and 5 days (Ohteki,T., et al. (1999) J. Exp. Med. 189:1981; Fukao, T., et al. (2000) Eur JImmunol 30:1453; Hochrein, H., et al. (2001) J Immunol 166:5448; Fukao,T., et al. (2001) J Immunol 166:4446; Fukao, T., et al. (2000) J Immunol164:64; Stober, D., et al. (2001) J Immunol 167:957). To investigatewhether T-bet plays a role during the early production of IFN-γ bysplenic DCs, a time course analysis of IFN-γ secretion during the firstthree days after stimulation with IL-12 alone, or in combination withIL-18, was performed and revealed an important role for T-bet (FIG.25C). In the absence of T-bet, splenic DCs displayed a 40 to 45%reduction in IFN-γ production during the first 24 hours, which continuedover the next 48 hours with a decrease in IFN-γ production that rangedbetween 60 to 70% (FIG. 25C). This impairment in IFN-γ production wasalso present in T-bet deficient DCs from BALB/c mice.

These studies demonstrate for the first time in the art that T-bet isexpressed in murine DCs and that its expression is controlled by IFN-γ.Thus T-bet is essential for the optimal secretion of IFN-γ by thisimportant cell type. This role is quite selective for IFN-γ in DCs asthe expression of other DC cytokines such IL-12, p35 and p40 subunits,TNFα, and IL-1 after stimulation with IL-12 and IL-18 or LPS were notaffected by the absence of T-bet.

Furthermore, while it is clear that Stat4 is absolutely required for theinitiation of IL-12-dependent production of IFN-γ in myeloid cells,T-bet also participates in the control of this cytokine. One possiblemechanistic scenario is that upon the interaction of a pathogen withToll receptor family members, the DC is stimulated to secrete IL-12 andIFN-γ, thereby activating both the Stat1 and Stat4 signaling pathways.Stat1 controls both the expression of T-bet and of Stat4 thussimultaneously maximizing the production of IFN-γ.

In conclusion, these studies demonstrate that T-bet influences thegeneration of Type I immunity not only by controlling Th1 lineagecommitment in the adaptive immune system but also by a direct influenceon the transcription of the IFN-γ gene in dendritic cells. T-bet istherefore a transcription factor that may be a molecular bridge betweenthe innate and adaptive immune systems.

Example 24 The Role of T-Bet in Mouse Models of Mycobacteriumtuberculosis

Susceptibility to Mycobacterium tuberculosis and Francisella tularensisvaries considerably in inbred mouse strains (ranging from highlysusceptible to intermediate to relatively resistant). Transgenic miceoverexpressing T-bet were produced and characterized on the C57BL6background, and have been backcrossed with the T-bet-deficient strainseveral generations onto the relatively resistant strain, C57BL/6, tobest assess its function in models of infection. The mechanism by whichT-bet provides resistance to both avirulent and virulent bacterialstrains was explored by taking advantage of T-bet overexpressor anddeficient mice that lack various cell populations e.g., CD4 or CD8 Tcells, NK and dendritic cells. The T-bet knock out and overexpressorstrains were backcrossed extensively onto both C57BL/6 and BALB/cstrains.

To test the function of T-bet in infection with Mycobacteriumtuberculosis, two groups of mice, T-bet knock out and wildtypelittermates, were infected intravenously with the standard dose ofvirulent MTB (100,000 CFU) and their survival was monitored. The resultswere as follows. Time to death (TTD) in days (±SD): T-bet knock out (4mice)=41 (±0.8), wild type (3 mice)=106 (±36), p<0.02. For comparison,TTD of a very susceptible strain of mice (C3HeB/FeJ) in thatexperiment=26(±1.2) and TTD of HcB15 (another susceptible strain)=34(±2.4). For further comparison, the most highly susceptible strain ofmice yet identified, IFNγ knock out mice on the B6 background, usuallydied within 15 days under the same experimental conditions.

At day 41 after infection, organs of 3 T-bet knock out mice wereharvested at the time of death or in a moribund state (one mouse) alongwith those of 1 wild type mouse (sacrificed), and histopathology, acidfast staining for bacteria and iNOS expression studies were carried out.A greater number of acid fast bacteria were found per macrophage in theT-bet knock out mice, however, neither pneumonia nor necrotic lesionsoften seen in the most susceptible strains of mice were observed.

In summary, the T-bet knock out mice were clearly more susceptible thantheir wild type littermates controls to infection with Mycobacteriumtuberculosis. The degree of susceptibility conferred by the absence ofT-bet is intermediate, in comparison to IFNγ deficient mice. Thesignificant degree of protection retained as compared to the IFNγ knockout may be due to residual IFNγ production by T-bet^(−/−) CD8 cells.

Example 25 Phosphorylation of T-Bet by Tec Kinases

The T-bet protein is phosphorylated. The kinase which phosphorylatesT-bet has been identified as a member of the Tec family of tyrosinekinases. ITK and Rlk/Txk are the predominant Tec family of tyrosinekinases expressed in T cells. FIG. 26 shows the conserved structure ofTec family members. The Tec family kinases have been shown to beimportant in cytokine secretion. Rlk/tkk is Thy specific and plays arole in the control of IFN-γ production. Itk−/− mice have reduced IL-4production while rlk/itk−/− mice demonstrated reduced Th1 and Th2cytokines. RIBP is an adapter protein that binds rlk and itk. RIBP−/−mice exhibit reduced IFN-γ and IL-2.

Both the ITK and Rlk/txk kinases have been found to phosphorylate T-betin vitro. The predicted tyrosine phosphorylation sites of human T-betare shown in FIG. 27. Modified forms of the T-bet protein were made andused as substrates in in vitro kinase assays (FIG. 28). Both ITK and Rlkphosphorylated N-terminal and C-terminal but not DNA-binding regions ofT-bet in in vitro kinase assays (FIG. 29).

Further indicating the importance of Tec kinases, diminished tyrosinephosphorylation of T-bet has been observed in ITK knock-out animals.T-bet was immunoprecipitated from T cells from B6, Balb/C, ITK knock outand RLK knock out animals. Western blots of the immunoprecipitates wereprobed with either anti-phosphotyrosine antibodies or anti-T-betantibodies. As shown in FIG. 30 although T-bet is present in T cellsfrom ITK knock out animals, tyrosine phosphorylation of the molecule isreduced. In contrast, T-bet was hyperphosphorylated in Rlk knockout Tcells indicating a role for Rlk in inhibiting T-bet tyrosinephosphorylation.

Example 26 Increased T_(R) Cells in the Absence of T-Bet

T regulatory (T_(R)) cells are essential for the induction of peripheraltolerance. Several types of T_(R) cells exist, including CD4(+) T cellswhich express CD25 constitutively and suppress immune responses viadirect cell-to-cell interactions, and type 1 T regulatory (T_(R) 1)cells, which function via secretion of interleukin (IL)-10 andtransforming growth factor (TGF)-beta. Suppression mediated byCD25(+)CD4(+) T cell clones is partially dependent on TGF-beta, but noton constitutive high expression of CD25. T_(R) cells are increased inthe absence of T-bet. The percentage of CD4+/CD25+ T cells wasdetermined by FACS analysis in T-bet+/+, T-bet+/−, and T-bet−/− mice. Asshown in FIG. 31, T-bet+/+ animals have approximately 3% T_(R) cells,T-bet+/− animals have approximately 20% T_(R) cells and T-bet−/− animalshave approximately 38% T_(R) cells.

Example 27 T-Bet Expression is Controlled Through the IFN-γ SignalingPathway

The following Materials and Methods were used in Example 26:

Mice, Antibodies and Cytokines

T-bet^(−/−) mice, backcrossed 5 generations onto the BALB/c background,were generated using techniques described herein. BALB/c, 129,STAT1^(−/−) and IFNγR1^(−/−) mice were purchased from Jacksonlaboratory.

Monoclonal anti-CD3 (2C11), anti-CD28, anti-IL4, anti-IFN-γ, anti-IL-12,and rmIFN-γ were all purchased from Pharmingen. Anti-IL-18, rmIL-18 werepurchased from Peprotech.

CD4⁺ T Cell Purification and Th Differentiation

Cervical, axillary, inguinal and popliteal lymph nodes were isolated andpassed through 40 μm filters. CD4⁺ T cells were purified by positiveselection using MACS purification (Miltenyi Biotech). For in vitroactivation 1×10⁶/ml T cells were resuspended in complete medium (RPMI1640 supplemented with 10% fetal calf serum (HyClone Laboratories),Hepes (100 mM), L-glutamine (2 mM), non-essential amino acids, sodiumpyruvate (1 mM), β-mercaptoethanol (50 μM), penicillin (50 units/ml),streptomycin (50 μg/ml) and stimulated with plate-bound 2 μg/ml anti-CD3and 2 μg/ml anti-CD28 for 3 days in the presence of 100 U/ml rhIL-2.Cells were then split 1:4 in complete medium and cultured for 4 days inthe presence of 100 U/ml rhIL2. To induce Th1 and Th2 differentiation asindicated in the figure legends the above cultures included (10 ng/mlrIL-12 and 10 μg/ml anti-IL4) for Th1 cell development or (10 ng/mlrIL4, 10 μg/ml anti-IFN-γ, and 10 μg/ml anti-IL-12) for Th2 celldevelopment.

ELISA and Intracellular Cytokine Staining

On day 7 after primary stimulation, 1×10⁶ cells were restimulated with 2g/ml plate bound anti-CD3. Supernatants were harvested after 24 hoursfor ELISA. Cytokines were captured during overnight incubation at 4° C.with 2 μg/ml plate bound anti-IFN-γ, anti-IL-4 and anti-IL-5 antibodies(Pharmingen), then sequentially labeled with secondary biotinylatedantibodies (Pharmingen) and alkaline phosphatase conjugated avidin(Sigma). Following incubation in phosphatase substrate (Sigma), cytokinelevels were measured against standards (Peprotech) at 405 nm. Forintracellular cytokine staining, cells were restimulated with PMA (50ng/ml) and ionomycin (1 μM) for 6 hours with the addition of monensin (3μM) for the last 3 hours. Cells were then washed in PBS, fixed in 4%paraformadehyde, and permeabilized in 0.1% saponin/1% FBS/PBS. Stainingwas performed with PE-conjugated anti-IFN-γ (Pharmingen) andFITC-conjugated anti-CD4 and analyzed by flow cytometry using a FACSCalibur as described (Szabo, S. et al. 2000 Cell 100: 655-69).

Real Time Transcript Quantification

CD4⁺ T cells were isolated and stimulated as described above in thepresence of indicated cytokines IL-12 (10 ng/ml) IFN-γ (500 U/ml) IL-4(10 ng/ml) or cytokine neutralizing antibodies (10 μg/ml). Cells, 5×10⁶per reaction, were stimulated in 2 ml medium and harvested at 3, 6 and24 hours. Total RNA was isolated using RNeasy (Qiagen) and subjected toreverse transcription using the Superscript first strand synthesissystem (Invitrogen). T-bet and IFN-γ transcripts were quantified usingan ABI 7700 Sequence Detector, and plotted relative to the housekeepinggene GAPDH. Primers and probes used were as follows: IFN-γ forward5′TCAAGTGGCATAGATGTGGAAGAA3′(SEQ ID NO:6), IFN-γ reverse5′TGGCTCTGCAGGATTTTCATG3′(SEQ ID NO:7), IFN-γ probe5′TCACCATCCTTTTGCCAGTTCCTCCAG3′(SEQ ID NO:8), T-bet forward5′CAACAACCCCTTTGCCAAAG3′(SEQ ID NO:9), T-bet reverse5′TCCCCCAAGCAGTTGACAGT3′(SEQ ID NO:10), T-bet probe5′CCGGGAGAACTTTGAGTCCATGTACGC3′(SEQ ID NO:11), GAPDH forward5′TTCACCACCATGGAGAAGGC3′(SEQ ID NO:12), GAPDH reverse5′GGCATGGACTGTGGTCATGA3′(SEQ ID NO:13), GAPDH probe5′TGCATCCTGCACCACCAACTGCTTAG3′(SEQ ID NO:14).

(Cytokine, 11(4):305-312; Immunity, 14:205-215).

Northern and Western Blot Analysis

Total RNA was isolated from CD4+ T cells using RNeasy (Qiagen) and 10 gof each sample separated on 1.2% agarose 6% formaldehyde gels,transferred onto GeneScreen membrane (NEN) in 20×SSC overnight andcovalently bound using a UV Stratalinker (Stratagene). Hybridization ofblots was carried out at 42° C. as described (Hodge, M. et al. 1996.Immunity 4: 1-20; Hodge, M. R., et al. 1996 Science 274: 1903-1905)using the radiolabeled T-bet, HPRT or GAPDH cDNA probes. Whole cellextracts were prepared as described (Gouilleux et al., (1994) EMBO13(18):4361-9). Extracts were separated by 10% PAGE followed byelectrotransfer to nitrocellulose membranes and probed with polyclonalantisera specific for T-bet followed by horseradishperoxidase-conjugated goat anti-rabbit IgG and enhancedchemiluminescence according to the manufacturer's instructions(Amersham).

T-bet expression is controlled primarily through the IFN-γR/STAT1signaling pathway, not the IL-12R/STAT4 pathway. This mode of signalingsets up a positive feedback loop between IFN-γ and T-bet during earlyTCR activation which propels the antigen activated Thp cell down the Th1pathway. Thus, the balance between IL-4 and IFN-γ during initial TCRstimulation has a major influence on the fate of a T helper cell. IL-12,while required at later stages of primary Th1 differentiation, does notappear to contribute to the initial stage of Th1 lineage induction.Taken together, our data suggest that there are discrete phases of Th1development in which STAT1, T-bet and STAT4 play distinct and criticalroles. T-bet directs Th1 lineage commitment through a self-reinforcingfeedback loop involving early IFN-γ production followed by proliferationand stabilization of committed Th1 cells by STAT4.

Several cytokines and cytokine receptors, such as IFN-γ, IL-18, andTCCR, have been implicated in alternative pathways of Th1differentiation or in augmentation of IL-12 induced Th1 development(Okamura, 1995 Nature. 378:88; Schmitt, 1994. Eur. J. Immunol. 24:793;Robinson D, et al. (1997) Immunity 7: 571-81; Macatonia S E, et al.(1993). Int. Immunol. 5: 1119-28; Chen Q, et al. (2000) Nature 407:916-20). IFN-γ plays an essential role during an immune response andexerts its effects by binding to the IFN-γ receptor, composed of twochains IFN-γR1 and R2, present on most cell types (Bach E A, et al.(1997) Annu Rev Immunol 15: 563-91; Boehm U, Klamp T, et al. (1997) AnnuRev Immunol 15: 749-95). However, the site and mechanism of IFN-γ'spromotion of Th1 differentiation remains obscure. This Example showsthat T-bet expression is controlled primarily through the IFN-γR/STAT1signaling pathway, not the IL-12R/STAT4 pathway, with the initial sourceof IFN-γ originating from the antigen activated CD4 T cell itself. ThusT-bet controls Th1 lineage commitment through a self-reinforcingfeedback loop involving early IFN-γ production.

Early T-Bet Expression Requires IFN-γ

Several signaling pathways have been implicated in promoting Th1differentiation and IFN-γ production. These include triggering of the Tcell receptor by MHC: antigen complexes and the cytokines IL-12, IL-18and IFN-γ. Given the central role of T-bet in Th1 differentiation wesought to determine which if any of these signals is responsible forinducing T-bet expression.

Purified CD4+ lymph node T cells from BALB/c mice were stimulated for 48hrs with 2 μg/ml plate bound anti-CD3 and anti-CD28 for 72 hours. Whereindicated IL-12 (20 ng/ml), IL-18 (20 ng/ml), IFN-γ (500 U/ml),anti-IFN-γ (10 μg/ml), anti-IL-12 (10 μg/ml), anti-IL-18 (10 μg/ml) wereadded. RNA was prepared and subjected to Northern blot analysis usingT-bet, IFN-γ GATA3 and HPRT cDNAs as probes. This analysis revealed thatsignaling through the TCR/CD28 pathway alone appeared sufficient toinduce T-bet expression with the inclusion of IL-12 slightly augmentingthis expression. However, the inclusion of antibodies to IFN-γ in theseCD4⁺ T cell cultures resulted in a dramatic loss of T-bet expression,while antibodies against both IL-12 and IL-18 failed to inhibit T-betinduction (FIG. 32). IFN-γ expression from these cells paralleled T-betexpression. In contrast to T-bet, the Th2 specific transcription factorGATA-3 was increased by anti-IFN-γ treatment and repressed by IL-12treatment. These data suggest that the expression of T-bet, as well asIFN-γ during primary stimulation is primarily dependent on IFN-γ. Themodest increase in T-bet following stimulation with IL-12 may besecondary to the capacity of IL-12 to augment IFN-γ expression (seebelow).

To assess the early kinetics of T-bet transcription, real-time PCR ofT-bet and IFN-γ mRNA was performed. As before, CD4⁺ lymph node T cellswere stimulated with anti-CD3/CD28 for 3, 6, and 24 hours in thepresence of IFN-γ, IL-12, IL-4 or neutralizing antibodies against thesecytokines. Consistent with the data above, T-bet transcripts wereinduced by signals involved in Th1 differentiation. Strikingly, cellsstimulated in the presence of antibody IFN-γ failed to producesignificant levels of T-bet. This inhibition was observed even in thepresence of the Th1 promoting cytokine IL-12. Interestingly at theseearly time points (3 and 6 hours after stimulation) the induction ofIFN-γ mRNA expression was unaffected by anti-IFN-γ treatment despite theinhibition of T-bet expression. Moreover, this early IFN-γ expressionwas unaffected by either Th1 or Th2 inducing conditions, a resultconsistent with recent studies by Grogen et al. (Immunity (2001) 14:205-15).

However, by 24 hours of stimulation in the presence of anti-IFN-γantibody, IFN-γ mRNA began to decrease and by 48 hours both IFN-γ andT-bet transcripts were absent (FIG. 32). Thus, the dependence on IFN-γfor T-bet expression and T-bet for sustained IFN-γ expression suggeststhat a self-reinforcing feedback mechanism may be occurring betweenIFN-γ and T-bet.

T-bet Expression is Downstream of Signals Emanating from the IFN-γReceptor

The importance of STAT family members in cytokine signal transductionand the differentiation of CD4⁺ T helper cells has been well documented(Murphy K M, et al. (2000) Annu. Rev. Immunol. 18: 451-94; O'Garra A, etal. (2000) Trends in Cell Biology 10: 542-50). Mice lacking STAT4 andSTAT6 fail to mount significant Th1 and Th2 responses, respectively(Kaplan M H, et al. (1996) Nature 382: 174-7; Thierfelder W E, et al.(1996) Nature 382: 171-4; Shimoda et al. (1996) Nature 380(6575):630-3;Takeda et al. (1996) Nature 380(6575) 627-30). In addition, IFN-γtranduces signals through STAT1, which plays a critical role in thegeneration of Th1 mediated immune responses. Thus, experiments wereconducted to definitively determine if these signaling molecules wereinvolved in the coordination of T-bet and IFN-γ expression. CD4⁺ lymphnode T cells were isolated from IFN-γ R^(−/−), STAT1^(−/−), andSTAT4^(−/−) mice and stimulated with plate bound anti-CD3/CD28+IL-2 (100U/ml) (neutral) or under Th1 (IL-12 and anti-IL-4) or Th2 (IL4,anti-IFN-γ, and anti-IL-12) inducing conditions for 72 h. RNA (on day 3)or whole cell lysates (on day 3 and day 6) were prepared and subjectedto either Northern blot analysis using T-bet, IFN-γ and HPRT cDNAs asprobes or were subjected to SDS-PAGE (10%) and subsequent Westernblotting. T-bet protein was detected using an anti-T-bet polyclonalantiserum followed by HRP conjugated goat anti-rabbit IgG (Santa Cruz)and ECL substrate (Amersham). In wildtype cells, as expected, T-bet andIFN-γ mRNA expression were restricted to T cells stimulated under Th1conditions. Interestingly, in all three deficient genotypes IFN-γ mRNAexpression was dramatically reduced when these cells were stimulatedunder Th1 conditions. In these same Th1 conditions T-bet expression wasmarkedly reduced in STAT1 and IFN-γ R1 deficient T cells yet STAT4deficient cells exhibited T-bet levels comparable to wildtype controls(FIG. 33). The same pattern was observed when T-bet protein expressionwas examined by Western blot analysis following 3 days of stimulation.Only after 6 days in culture did STAT4^(−/−) T cells begin to show areduction in T-bet expression. These data clearly demonstrate thatsignals emanating from the IFN-γ receptor via STAT1 are necessary forthe induction of T-bet expression, while signaling through the STAT4pathway is not required for this early T-bet induction. The latereduction in T-bet expression in STAT4 deficient T cells is likely dueto reduced levels of IFN-γ in these cultures. The reduced IFN-γ levelsin STAT4 deficient cells suggests that a key role for STAT4 may be tosustain high level IFN-γ production in the developing Th1 response.

Impaired IFN-γ Production in T-bet−/− and STAT1−/− CD4+ T Cells

Having established a role for IFN-γ and STAT1 in the induction of T-betexpression, the Th1 differentiation capacity and cytokine profiles ofcells lacking T-bet versus those lacking either STAT1 or STAT4 wascompared. Moreover, while numerous studies have demonstrated theintegral role of STAT4 in Th1 development (Murphy K M, et al. (2000)Annu. Rev. Immunol. 18: 451-94), a similar analysis of STAT1 has beenlacking. Primary cultures of CD4+ T-bet^(−/−), STAT1^(−/−) andSTAT4^(−/−) CD4⁺ lymph node T cells were isolated and stimulated withplate bound anti-CD3/CD28+IL-2 (neutral) or with the addition of Th1inducing conditions. Cultures were expanded on day 3 with additionalIL-2. On day 7 cells were washed, restimulated and cytokine productionwas assayed by ICC and ELISA. For ICC, cells were restimulated withPMA/ionomycin for 6 hrs with the addition of monensin during the last 3hrs. For ELISA, cells were restimulated with plate bound anti-CD3 for 24hours and culture supernatants were analyzed for IFN-γ. C. IncreasedIL-4 and IL-5 production from T-bet deficient T cells. CD4+ T cells fromT-bet^(−/−), STAT1^(−/−) and STAT4^(−/−) and wildtype controls wereisolated and activated under neutral, Th1 or Th2 conditions. On day 7cells were restimulated and assayed for IL-4 and IL-5 production byELISA. A comparison of these three genotypes clearly illustrates thefundamental role of T-bet and STAT1 in IFN-γ production and Th1development. Most dramatically, under neutral conditions, ICC stainingshowed very few IFN-γ producing cells in the T-bet^(−/−) and STAT1^(−/−)populations while there was no difference in the percentage ofIFN-γ-positive cells in the STAT4^(−/−) T cells as compared to wild-typecontrols (FIG. 34). Following stimulation under Th1 skewing conditions,each deficiency exhibited a decreased percentage of IFN-γ-positivecells, with the greatest reductions observed in T-bet^(−/−) andSTAT1^(−/−) T cells (FIG. 35A). ELISA measurement of IFN-γ levels inculture supernatants showed comparable results to the ICC staining.

Analysis of Th2 cytokine production in these cultures was performed tofurther characterize the developing Th phenotypes of T-bet^(−/−),STAT^(−/−) and STAT4^(−/−) T cells. Under Th2 conditions, each genotypeproduced IL-4 and IL-5 (as measured by ELISA) in quantities comparableto wildtype controls. T-bet deficient T cells produced markedly higherIL-4 and IL-5 under both neutral and Th1 conditions as compared towildtype controls. Additionally, in neutral and Th1 conditions, STAT1deficient T cells showed increased IL-4 production as compared towildtype. In contrast, STAT4 deficient cells showed no difference ineither IL-4 or IL-5 production as compared to wildtype cells in eitherneutral or Th1 conditions. Taken together, these studies demonstratethat an optimal Th1 response, characterized by abundant IFN-γ productionand suppression of Th2 cytokines, requires the presence of both T-betand STAT1.

IFN-γ was also measured in STAT4−/− and T-bet−/− double knock outanimals. As above, FIG. 35B shows that Th1 cells from STAT4 and T-betknock out animals exhibited reduced IFN-γ production, with the T-betknock out showing the greatest decrease. The STAT4/T-bet double knockout, however, showed an even greater reduction in IFN-γ production.

These data show that there are discrete phases of Th1 cell developmentin which STAT1, T-bet and STAT4 have distinct roles. IFN-γ/STAT1 is theprimary regulator of T-bet expression, not IL-12/STAT4. When IFN-γ wasneutralized during primary stimulation, T-bet expression was lost andcells failed to fully commit to the Th1 lineage. These studies wereconfirmed in experiments using STAT1^(−/−) and IFNγR1^(−/−) T cellswhich showed that T-bet expression was severely diminished, as wassubsequent production of IFN-γ.

Although Th1 development was greatly compromised in STAT4^(−/−) T cells,early expression of T-bet was not impaired. Initially, this observationseemed paradoxical given the central roles of both STAT4 and T-bet inTh1 differentiation. However, analysis of T-bet, STAT1 and STAT4deficient T cells stimulated under neutral (without the addition of Th1promoting stimuli) conditions could offer an explanation. While IFN-γproduction was essentially absent in both STAT1^(−/−) and T-bet^(−/−) Tcells, STAT4^(−/−) cells produced IFN-γ levels comparable to wildtypecontrols. This result suggests that STAT4 is not necessary for directlyinducing IFN-γ production. Thus, the central role of STAT4 in Th1development may be attributed to STAT4's ability to augment andstabilize the amount of IFN-γ produced during a Th1 response.

Locksley and colleagues have shown that immediately following TCR/CD28engagement, naïve Thp cells express both IL-4 and IFN-γ transcripts,independent of STAT4 and STAT6 (Grogan J L, et al. (2001) Immunity 14:205-15). This example corroborates this and demonstrates that initialIFN-γ expression is also independent of T-bet. Subsequently, withinhours, the Thp cell enters a second, cytokine dependent, phase ofcommitment. Here, we find that T-bet expression is necessary to supportTh1 development, and relies on signals emanating from the IFN-γR/STAT1signaling pathway. During this secondary phase, while IFN-γ productionis maintained, IL-4 expression is extinguished in developing Th1 cells(Grogan J L, et al. (2001) Immunity 14: 205-15)

Without wishing to be bound by any particular mechanism, T-bet maypotentiate IFN-γ production at this stage, not by initiating chromatinremodeling of the IFN-γ allele, but perhaps by stabilizing the alreadyopen IFN-γ locus. T-bet may also induce expression of the IL-12Rα2chain, allowing Th1 development to enter a third, IL-12/STAT4 dependentstage. Here IL-12 optimizes the level of IFN-γ produced and/or serves asa growth factor for committed Th1 cells (i.e. those expressing afunctional IL-12R complex).

Taken together, these data suggest that this early cytokine expressionmay be the stochastic aspect of Th cell differentiation. Thus, whileeach Thp cell expresses both IL-4 and IFN-γ, they may not be expressedat the exact same time. During the initial burst of cytokinetranscription, it may be arbitrary as to which cytokine gene isexpressed first and which of the two alleles is expressed first.

This model proposes that the outcome of this first stochastic step playsan important role in directing the fate of an individual Thp cell due toa self-reinforcing feedback mechanism involving IFN-γ and T-bet or IL-4and GATA3. For example, a naïve Thp cell is stimulated via the TCR/CD28and may randomly initiate IFN-γ expression. The secreted IFN-γ then actson the IFN-γR present on that naïve Thp cell to induce T-bet which inturns supports the expression of more IFN-γ followed by more T-bet. Atsome point the IL-4 gene is randomly expressed and the secreted IL-4binds to the IL-4R expressed on that same naïve cell which induces GATA3expression. However, at this point GATA3 may not be able to overcome thegradient of T-bet initially established by the first stochastic act ofIFN-γ expression. At this stage T-bet may inhibit IL-4 and IL-5expression by suppressing GATA3 expression. Additionally in thedeveloping response the IL-12Rα2 chain is expressed allowing IL-12 toact as a growth factor for the developing Th1 cell and/or act toincrease IFN-γ levels. Thus, early IFN-γ and T-bet act to initiate andpotentiate Th1 development, followed by signals transduced by STAT4 thatenhance and stabilize Th1 lineage commitment.

Nonetheless, our studies argue against a purely selective model. Underthis hypothesis, Th cell development is a process solely determined bygrowth signals delivered by cytokines to T cells that have initiallyundergone a cytokine-independent stochastic process of Th1 or Th2differentiation. If this model were to be correct, one would expect thatT cells lacking STAT1 would retain the ability to produce IFN-γ.However, in neutral culture conditions IFN-γ producing STAT1^(−/−) cellsare essentially absent. Moreover, IFN-γ historically is known for itsanti-proliferative capacity and STAT1^(−/−) cells have increased growthrates compared to wildtype cells (Durbin J E, et al. (1996) Cell 84:443-50; Meraz M A, et al. (1996) Cell 84: 431-42)

Thus one would predict based on the selection model that there should bemore IFN-γ producing STAT1^(−/−) cells than wildtype cells. Even in Th1culture conditions there is a severe reduction in the ability of theSTAT1^(−/−) Th cells to produce IFN-γ and differentiate toward the Th1phenotype. Thus, while these data suggest that the IL-12/STAT4 pathwayis not involved in the initial promotion of Th1 differentiation, itappears that signals transduced by IFN-γ/STAT1, via an instructivemechanism, are required for the efficient generation of IFN-γ and theTh1 response.

Signaling through the IFN-γ receptor pathway appears to be the primaryinducer of T-bet. T-bet transcripts were completely lacking inSTAT1^(−/−) T cells stimulated under neutral conditions. TCR stimulationalone was not sufficient to induce T-bet expression nor did it induceT-bet autoregulation. This finding contrasts with prior studies thatsupported an autoregulatory mechanism for T-bet in experiments measuringendogenous T-bet transcripts when ectopic T-bet was retrovirallyintroduced into STAT4^(−/−) T cells (Mullen A C, et al. (2001) Science292: 1907-10). However, retrovirally expressed T-bet has been shown topotently induce endogenous IFN-γ production (Szabo et al. (2000) Cell100:655-69; Mullen A C, et al. (2001) Science 292: 1907-10) and theIFN-γ produced would then be able to bind IFN-γ receptors present on theSTAT4^(−/−) T cells and induce endogenous T-bet. Thus, we find that anintact IFN-γ R1/STAT1 signaling pathway is essential to obtain highlevel T-bet expression. However, despite the striking reduction of T-bettranscripts in STAT1^(−/−) and IFNγR1^(−/−) T cells developing under Th1conditions, a low level of T-bet transcripts remained. These resultssuggest that there are yet to be discovered IFN-γ independentsignals/mechanisms that can induce T-bet and Th1 differentiation.

The data presented in combination with previous T-bet expression studiessuggests the following model of early T-helper cell differentiation.Initial production of the signature Th1 and Th2 cytokines, IFN-γ andIL-4 respectively, are driven by TCR mediated signals. Commitment toeither developmental pathway depends on the cytokine mediated inductionof the transcription factors T-bet and GATA3 and subsequentstabilization of each lineage through multiple components, i.e. STATs.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for identifying a compound that modulates the activity of aT-bet polypeptide, comprising providing a recombinant cell thatcomprises an expression vector encoding the T-bet polypeptide, whereinthe T-bet polypeptide binds a consensus T-box site in DNA and inducesIFN-γ production in CD4+ cells, and wherein said expression vectorcomprises: i) a nucleic acid molecule which hybridizes to the fulllength complement of the nucleic acid molecule set forth in SEQ ID NO:1in 6×SSC at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDSat 65° C.; or ii) a nucleic acid molecule which hybridizes to the fulllength complement of the nucleic acid molecule set forth in SEQ ID NO:3in 6×SSC at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDSat 65° C.; or iii) a nucleic acid molecule encoding a polypeptide withat least 90% amino acid identity to SEQ ID NO:2; or iv) a nucleic acidmolecule encoding a polypeptide with at least 90% amino acid identity toSEQ ID NO:4; said recombinant cell further comprising: v) a nucleic acidmolecule comprising a Tbox binding site to which the T-bet polypeptidebinds; contacting the recombinant cell with a test compound; andevaluating the effect of the test compound by determining the binding ofthe T-bet polypeptide to the nucleic acid molecule comprising the T-boxbinding site in the presence and absence of the test compound, tothereby identify a compound that modulates the activity of the T-betpolypeptide.
 2. The method of claim 1, wherein the expression vectorcomprises a nucleic molecule encoding the polypeptide of SEQ ID NO: 4.3. The method of claim 1, wherein the expression vector comprises anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1.4. The method of claim 1, wherein the expression vector comprises anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3.5. The method of claim 1, wherein the nucleic acid molecule of v) is anIL-2 promoter.
 6. The method of claim 2, wherein the nucleic acidmolecule of v) is an IFN-γ promoter.
 7. The method of claim 1, furthercomprising determining the effect of the test compound on an immuneresponse in a non-human animal, comprising administering the testcompound to the animal and determining the effect of test compound on animmune response, to thereby identify a compound that modulates an immuneresponse.
 8. The method of claim 1, wherein the activity of thepolypeptide is enhanced.
 9. The method of claim 1, wherein the activityof the polypeptide is inhibited.
 10. The method of claim 1, wherein thetest compound is selected from the group consisting of: nucleic acidmolecules, peptides, and small molecules.
 11. The method of claim 1,further comprising determining the effect of the test compound on T or Bcell responsiveness in a cell-based assay.
 12. The method of claim 11,wherein T or B cell responsiveness is determined by measuring and effectof the test compound on an outcome selected from the group consistingof: IgG class switching, Th1 cell development, Th2 cell development, andB lymphocyte function.
 13. The method of claim 1, wherein the nucleicacid molecule of v) comprises a reporter gene operably linked to a T-betresponsive promoter comprising the T-box binding site, and the abilityof the T-bet polypeptide to bind to the promoter is determined byevaluating the expression of the reporter gene in the presence andabsence of the test compound.
 14. The method of claim 13, wherein thereporter gene is selected from the group consisting of IL-2 and IFN-γ.15. The method of claim 14, wherein the reporter gene is IFN-γ.
 16. Themethod of claim 13, wherein the reporter gene is selected from the groupconsisting of genes that encode: chloramphenicol acetyltransferase,beta-galactosidase, alkaline phosphatase and luciferase.
 17. The methodof claim 1, wherein the expression vector comprises a nucleic moleculeencoding the polypeptide of SEQ ID NO:
 2. 18. The method of claim 17,wherein the nucleic acid molecule of v) is an IL-2 promoter.
 19. Themethod of claim 17, wherein the nucleic acid molecule of v) is an IFN-γpromoter.
 20. The method of claim 17, further comprising determining theeffect of the test compound on an immune response in a non-human animal,comprising administering the test compound to the animal and determiningthe effect of test compound on an immune response, to thereby identify acompound that modulates an immune response.
 21. The method of claim 17,wherein the activity of the polypeptide is enhanced.
 22. The method ofclaim 17, wherein the activity of the polypeptide is inhibited.
 23. Themethod of claim 17, wherein the test compound is selected from the groupconsisting of: nucleic acid molecules, peptides, and small molecules.24. The method of claim 17, further comprising determining the effect ofthe test compound on T or B cell responsiveness in a cell-based assay.25. The method of claim 24, wherein T or B cell responsiveness isdetermined by measuring and effect of the test compound on an outcomeselected from the group consisting of: IgG class switching, Th1 celldevelopment, Th2 cell development, and B lymphocyte function.
 26. Themethod of claim 17, wherein the nucleic acid molecule of v) comprises areporter gene operably linked to a T-bet responsive promoter comprisingthe T-box binding site, and the ability of the T-bet polypeptide to bindto the promoter is determined by evaluating the expression of thereporter gene in the presence and absence of the test compound.
 27. Themethod of claim 26, wherein the reporter gene is selected from the groupconsisting of IL-2 and IFN-γ.
 28. The method of claim 27, wherein thereporter gene is IFN-γ.
 29. The method of claim 26, wherein the reportergene is selected from the group consisting of genes that encode:chloramphenicol acetyltransferase, beta-galactosidase, alkalinephosphatase and luciferase.
 30. A method for identifying a compound thatmodulates the activity of a T-bet polypeptide, comprising providing arecombinant cell that comprises an expression vector encoding the T-betpolypeptide, wherein the T-bet polypeptide binds a consensus T-box sitein DNA and induces IFN-γ production in CD4+ cells, and wherein saidexpression vector comprises: i) a nucleic acid molecule which hybridizesto the full length complement of the nucleic acid molecule set forth inSEQ ID NO:1 in 6×SSC at 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; or ii) a nucleic acid molecule whichhybridizes to the full length complement of the nucleic acid moleculeset forth in SEQ ID NO:3 in 6×SSC at 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 65° C.; or iii) a nucleic acid moleculeencoding a polypeptide with at least 90% amino acid identity to SEQ IDNO:2; or iv) a nucleic acid molecule encoding a polypeptide with atleast 90% amino acid identity to SEQ ID NO:4; said recombinant cellfurther comprising: v) a tec kinase molecule to which the T-betpolypeptide binds; contacting the recombinant cell with a test compound;and evaluating the effect of the test compound by determining thebinding of the T-bet polypeptide to the tec kinase molecule in thepresence and absence of the test compound, to thereby identify acompound that modulates the activity of the T-bet polypeptide.
 31. Themethod of claim 30, wherein the tec kinase is selected from the groupconsisting of ITK and rlk.